JP2016501195A - Oligonucleotide conjugate - Google Patents

Abstract

The present invention relates to the field of oligonucleotide pharmacology, specifically to covalently conjugate, targeting, or blocking groups to enhance the properties of the oligonucleotide, for example to improve the therapeutic index. It relates to the use of attached cleavable regions, for example phosphodiester regions.

Description

FIELD OF THE INVENTION The present invention relates to the field of oligonucleotide pharmacotherapeutics, in particular of conjugates, targeting groups or blocking groups to enhance the properties of oligonucleotides, for example to improve therapeutic indices. Regarding use.

Related Matters This application claims priority under EP12192773.5, EP13153296.2, EP13157237.2, and EP13174092.0, which are incorporated herein by reference.

Background Oligonucleotide conjugates have been extensively evaluated for use in siRNA and are considered essential to obtain sufficient in vivo efficacy. For example, see WO2004 / 044141 (Patent Document 1) which refers to a modified oligomeric compound that regulates gene expression via the RNA interference pathway. The oligomeric compound comprises one or more conjugate moieties that can modify or enhance the pharmacokinetic and pharmacodynamic properties of the attached oligomeric compound.

  In contrast, single stranded antisense oligonucleotides are typically administered therapeutically without conjugation or formulation. The primary target tissues of antisense oligonucleotides are the liver and kidneys, but a wide range of other tissues, including lymph nodes, spleen, and bone marrow, are also reachable by antisense modalities.

  WO2008 / 113832 (Patent Document 2) discloses an LNA phosphorothioate gapmer (gapmer) oligonucleotide in which adjacent regions contain at least one phosphodiester between or adjacent to LNA nucleosides. Oligomers were preferentially targeted to the kidney.

  WO2004 / 087931 (Patent Document 3) refers to an oligonucleotide containing a hydrophilic polymer (PEG) conjugate cleavable by acid.

  WO2005 / 086775 refers to target-specific delivery of therapeutic agents to specific organs using therapeutic chemical moieties, cleavable linkers, and label domains. The cleavable linker can be, for example, a disulfide group, a peptide, or an oligonucleotide domain cleavable by a restriction enzyme.

  WO2009 / 126933 (Patent Document 5) refers to specific delivery of siRNA nucleic acids by combining a targeting ligand with an endosomolytic component.

  WO2011 / 126937 (patent document 6) refers to target-specific intracellular delivery of oligonucleotides via conjugation with small molecule ligands.

  WO2009 / 025669 (Patent Document 7) refers to a polymer (polyethylene glycol) linker containing a pyridyl disulfide moiety. See also Zhao et al., Bioconjugate Chem. 2005 16 758-766 (Non-Patent Document 1).

  Chaltin et al., Bioconjugate Chem. 2005 16 827-836 was modified by cholesterol, which was used to incorporate antisense oligonucleotides into cationic liposomes to create dendrimer delivery systems. Monomer, dimer, and tetramer oligonucleotides are reported. Cholesterol is conjugated to the oligonucleotide via a lysine linker.

  Other non-cleavable cholesterol conjugates have been used to target siRNA and antagomir to the liver. For example, see Soutscheck et al., Nature 2004 vol. 432 173-178 (Non-Patent Document 3) and Krutzfeldt et al., Nature 2005 vol 438, 685-689 (Non-Patent Document 4). The use of cholesterol as a liver targeting entity for partially phosphorothioated siRNA and antagomir was found to be essential for in vivo activity.

  The present invention is based on the use of a homing device linked to an oligonucleotide by a short region of a nuclease labile nucleoside, such as a DNA nucleoside or an RNA nucleoside linked by a phosphodiester, to make a highly effective target specific of the oligonucleotide. Based on the discovery that delivery is achieved.

WO2004 / 044141 WO2008 / 113832 WO2004 / 087931 WO2005 / 086775 WO2009 / 126933 WO2011 / 126937 WO2009 / 025669

Zhao et al., Bioconjugate Chem. 2005 16 758-766 Chaltin et al., Bioconjugate Chem. 2005 16 827-836 Soutscheck et al., Nature 2004 vol.432 173-178 Krutzfeldt et al., Nature 2005 vol 438, 685-689

The present invention provides oligomeric compounds comprising the following three regions:
(I) a first region (region A) comprising 7 to 26 contiguous nucleotides;
(Ii) a second region comprising 1-10 nucleotides (region B) covalently linked to the 5 ′ nucleotide or 3 ′ nucleotide of the first region via, for example, an internucleoside linking group, such as a phosphodiester linkage. ),here,
(A) the internucleoside linkage between the first region and the second region is a phosphodiester linkage, and the nucleoside of the second region adjacent [eg directly] to the first region is either DNA or RNA; And / or (b) a DNA or RNA nucleoside in which at least one nucleoside of the second region is linked by a phosphodiester;
(Iii) A third region (C) comprising a conjugate moiety, targeting moiety, reactive group, activating group, or blocking moiety and covalently linked to the second region.

  In some embodiments, region A and region B form a single contiguous nucleotide sequence that is 8 to 35 nucleotides in length.

  In some aspects, the internucleoside linkage between the first region and the second region can be considered part of the second region.

  In some embodiments, there is a phosphorus-containing linking group between the second region and the third region. The phosphorus linking group can be, for example, a phosphate (phosphodiester) group, a phosphorothioate group, a phosphorodithioate group, or a boranophosphate group. In some embodiments, the phosphorus-containing linking group is located between the second region and the linker region attached to the third region. In some embodiments, the phosphate group is a phosphodiester.

  Thus, in some aspects, the oligomeric compound comprises at least two phosphodiester groups, at least one is as described above of the invention, and the others are between the second region and the third region, Optionally, located between the linker group and the second region.

  In some embodiments, the third region is an activating group, such as an activating group for use in conjugation. In this regard, the present invention also provides an intermediate suitable for conjugation, such as conjugation with region A and region B and an activated oligomer comprising an activating group, for example a subsequent third region. .

  In some embodiments, the third region is a reactive group, eg, a reactive group for use in conjugation. In this regard, the present invention also provides intermediates suitable for conjugation, such as conjugation with regions A and B and an oligomer comprising a reactive group, for example a subsequent third region. The reactive group may in some embodiments comprise an amine group or an alcohol group, such as an amine group.

  In some embodiments, region A is an internucleoside linkage other than a phosphodiester, eg, (optionally) an internucleoside linkage selected from the group consisting of phosphorothioate, phosphodithioate, and boranophosphate, and methylphosphonate. For example, phosphorothioate at least 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, region A includes at least one phosphorothioate linkage. In some embodiments, at least 50% of the internucleoside linkages, such as at least 75%, such as at least 90%, eg, all internucleoside linkages within region A are other than phosphodiester, eg, phosphorothioate linkages. In some embodiments, all internucleoside linkages within region A are other than phosphodiesters.

  In some embodiments, the oligomeric compound comprises an antisense oligonucleotide, eg, an antisense oligonucleotide conjugate. The antisense oligonucleotide may be or include a first region, optionally a first region and a second region. In this regard, in some embodiments, region B may form part of a contiguous nucleobase sequence that is complementary to a (nucleic acid) target. In other embodiments, region B may lack complementarity with the target.

  In other words, in some embodiments, the present invention has at least one terminal (5 ′ and / or 3 ′) DNA or RNA nucleoside linked to the adjacent nucleoside of the oligonucleotide via a phosphodiester linkage. Provided are oligonucleotides (eg, antisense oligonucleotides) linked by non-phosphodiesters, eg linked by phosphorothioates. The terminal DNA or RNA nucleoside is further covalently linked to the conjugate, targeting or blocking moiety, optionally via a linker moiety.

  The present invention provides a pharmaceutical composition comprising an oligomeric compound of the present invention and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.

  The present invention provides an oligomeric compound according to the present invention for use in the inhibition of nucleic acid targets in cells. In some embodiments, the use is in vitro. In some embodiments, the use is in vivo.

  The present invention provides an oligomeric compound of the invention for use in medicine, for example for use as a medicament.

  The present invention provides an oligomeric compound of the invention for use in the treatment of a medical disease or disorder.

  The present invention provides the use of an oligomeric compound of the invention for the preparation of a medicament for the treatment of a disease or disorder, eg a metabolic disease or disorder.

The present invention provides a method for synthesizing (or producing) an oligomeric compound, for example, an oligomeric compound of the present invention. This law
(A) One of the following [third area]:
(I) an optional linker group (-Y-),
(Ii) A group X (X-) or -YX group containing a conjugate, targeting group, blocking group, reactive group [eg, amine or alcohol], or a group selected from the group consisting of an activating group is attached. Providing a [solid phase] oligonucleotide synthesis support, and (b) a step of region B followed by a [sequential] oligonucleotide synthesis of region A, and / or (c) the first region (A) and Comprising the step of [sequential] oligonucleotide synthesis of the second region (B), and following the synthesis step,
(D) the third region [including phosphoramidite],
(I) an optional linker group (-Y-),
(Ii) a group X (X-) comprising a conjugate, targeting group, blocking group, reactive group [eg amine or alcohol], or a group selected from the group consisting of activating groups, or an optional -YX group Followed by the process of adding
(E) [solid phase] cleaving the oligomeric compound from the support, optionally,
(F) the third group is an activating group and activates the activating group to produce a reactive group, followed by a conjugate group, blocking group, or targeting group, optionally via a linker group (Y) Step of adding to the reactive group:
(G) the third region is a reactive group, optionally adding a conjugate group, a blocking group, or a targeting group to the reactive group via a linker group (Y);
(H) the third region is a linker group (Y) and further comprises a further step selected from the step of adding a conjugate group, blocking group, or targeting group to the linker group (Y);
Step (f), (g), or (h) is performed either before or after cleavage of the oligomeric compound from the oligonucleotide synthesis support. In some embodiments, the method can be performed using standard phosphoramidite chemistry, so that region X and / or region X or regions X and Y are prepared as phosphoramidites prior to incorporation into the oligomer. Can be done. Reference is made to FIGS. 5-10 illustrating non-limiting aspects of the method of the present invention.

  The present invention provides a method of synthesizing (or producing) an oligomeric compound, eg, an oligomeric compound of the present invention, which comprises a [sequential] oligo in the first region (A) and optionally the second region (B). Includes a step of nucleotide synthesis, and after the synthesis step, region X (also referred to as region C), which is the third region [including phosphoramidite], or Y, eg, conjugate, targeting group, blocking group, functional group, reactive group [For example, amine or alcohol], or a region containing a group selected from the group consisting of an activating group (X), or a step of adding a -YX group, followed by cleavage of the oligomeric compound from the [solid phase] support The process of carrying out is included.

  However, it will be appreciated that region X or XY may be added after cleavage from the solid support. Alternatively, the synthetic method comprises synthesizing a first region (A), and optionally a second region (B), followed by cleaving the oligomer from the support, and a third region, eg, an X group or Subsequent steps of adding XY groups to the oligomer may be included. The addition of the third region can be achieved, for example, by adding aminophosphoramidite units in the final step of oligomer synthesis (on the support). It can be used for conjugation with the X or X—Y group, optionally after cleavage from the support, via an activating group on the X or Y group (if present). In embodiments where the cleavable linker is not a nucleotide region, region B may form part of region X (also referred to as region C) or non-nucleotide cleavable linker, eg peptide It can be a linker.

  In some embodiments of the method, region X (eg, C) or (XY), eg, a conjugate (eg, a GalNAc conjugate) comprises an activating group (an activated functional group), and in the method of synthesis The activated conjugate (or region x or XY) is added to the oligomer linked by the first region and the second region, eg amino. Amino groups can be added to the oligomer by standard phosphoramidite chemistry, for example, as a final step in oligomer synthesis (typically resulting in an amino group at the 5 ′ end of the oligomer). For example, in the final step of oligonucleotide synthesis, a protected amino-alkyl phosphoramidite, such as TFA-amino C6 phosphoramidite (6- (trifluoroacetylamino) -hexyl- (2-cyanoethyl)-(N, N- Diisopropyl) -phosphoramidite) is used. Region X (or region C referred to herein), eg, a conjugate (eg, a GalNac conjugate) can be activated via the NHS ester method, and then an amino linked oligomer is added. . For example, N-hydroxysuccinimide (NHS) can be used as an activating group for region X (or region C), eg, a conjugate, eg, a GalNac conjugate moiety. The present invention provides an oligomer prepared by the method of the present invention.

  In some embodiments, region X and / or region X or regions X and Y are linked via phosphate nucleoside linkages, such as those described herein, such as phosphodiesters or phosphorothioates, or alternative groups. , May be covalently joined (linked) to region B via, for example, a triazole group.

  The present invention provides a method of treating a disease or disorder in a subject in need of treatment comprising the step of administering to the subject a pharmaceutical composition comprising an oligomeric compound of the present invention in a therapeutically effective amount.

  The present invention comprises the step of administering the oligomeric compound according to the present invention to a cell expressing the target gene appropriately in an amount effective to reduce the expression of the target gene in the cell. Methods of inhibiting expression are provided. In some embodiments, the method is performed in vitro, ie, outside the organism, but may be performed in (eg, ex vivo) cells or tissues. In some embodiments, the method is in vivo.

  The present invention also provides an LNA oligomer comprising a continuous region of nucleosides linked by 8-24 phosphorothioates and further comprising 1-6 DNA nucleosides continuous with the LNA oligomer. Internucleoside linkages between and / or adjacent to DNA nucleosides are physiologically unstable, eg, phosphodiester linkages. Such LNA oligomers may take the form of conjugates described herein, or may be intermediates used, for example, in subsequent conjugation steps. When conjugated, the conjugate may be or include, for example, a sterol, such as cholesterol or tocopherol, or a (non-nucleotide) carbohydrate, such as a GalNac conjugate, such as a GalNac cluster, For example, it may be or include TriGalNac, or other conjugates described herein.

  The present invention relates to an LNA antisense oligomer (herein described) comprising an antisense oligomer and an asialoglycoprotein receptor targeting moiety conjugate moiety, eg, a GalNAc moiety, that can form part of an additional region (referred to as region C). Which can be referred to as region A) in the document. LNA antisense oligomers can be 7-30 nucleosides long, eg, 8-26 nucleosides long, and include at least one LNA unit (nucleoside).

  The present invention provides LNA antisense oligomers covalently joined (eg, linked) to (non-nucleoside) carbohydrate moieties, eg, carbohydrate conjugate moieties. In some embodiments, the carbohydrate moiety is not a linear carbohydrate polymer. However, the carbohydrate moiety may be multivalent, e.g., divalent, trivalent, tetravalent, or four identical or non-identical carbohydrate moieties are optionally covalently attached to the oligomer via a linker. May be joined together.

  The present invention provides an LNA antisense oligomer (conjugate) comprising an antisense oligomer and a conjugate moiety comprising a carbohydrate, eg, a carbohydrate conjugate moiety.

  The present invention provides a pharmaceutical composition comprising the LNA oligomeric compound of the present invention and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.

  The present invention provides an oligomeric compound according to the present invention for use in the inhibition of nucleic acid targets in cells. In some embodiments, the use is in vitro. In some embodiments, the use is in vivo.

  The present invention provides an oligomeric compound of the invention for use in medicine, for example for use as a medicament.

  The present invention provides an oligomeric compound of the invention for use in the treatment of a medical disease or disorder.

  The present invention provides the use of an oligomeric compound of the invention for the preparation of a medicament for the treatment of a disease or disorder, eg a metabolic disease or disorder.

Non-limiting illustration of an oligomer of the invention attached to an activating group (ie a protected reactive group-as a third region). The internucleoside linkage L can be, for example, a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate or methylphosphonate, such as a phosphodiester. PO is a phosphodiester bond. Compound (a) has region B having a single DNA or RNA, and the bond between the second region and the first region is PO. Compound (b) has two DNA / RNA (eg, DNA) nucleosides linked by phosphodiester bonds. Compound (c) has three DNA / RNA (eg, DNA) nucleosides linked by phosphodiester bonds. In some embodiments, region B can be further extended by additional phosphodiester DNA / RNA (eg, DNA nucleoside). The activating group is illustrated on the left side of each compound, and a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, such as a phosphonucleoside linking group, or in some embodiments, a triazole linkage. And optionally linked to the terminal nucleoside of region B. Compounds (d), (e), and (f) further comprise a linker (Y) between region B and the activating group, where region Y is, for example, phosphodiester, phosphorothioate, phosphorodithioate, borano A phosphonucleoside linking group, such as phosphate, or methylphosphonate, or in some embodiments, can be linked to region B via a triazole bond. Compounds equivalent to those shown in FIG. 1; however, reactive groups are used instead of activating groups. The reactive group can be the result of activation (eg, deprotection) of the activating group in some embodiments. The reactive group can be an amine or an alcohol in non-limiting examples. Non-limiting illustration of compounds of the invention. Same terminology as in Figure 1. X may in some embodiments be a conjugate, such as a lipophilic conjugate such as cholesterol or another conjugate such as those described herein. In addition or alternatively, X can be a targeting group or a blocking group. In some aspects, X can be an activating group (see FIG. 1) or a reactive group (see FIG. 2). X may be covalently attached to region B via a phosphonucleoside linking group, such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, or an alternative bond, For example, they may be linked via a triazole bond (see L in compounds (d), (e), and (f)). FIG. 3 is a non-limiting illustration of a compound of the invention comprising an optional linker between the third region (X) and the second region (region B). Same terminology as in Figure 1. Suitable linkers are disclosed herein and include, for example, alkyl linkers, such as C6 linkers. In compounds A, B, and C, the linker between X and region B is a region B via a phosphonucleoside linking group, such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate. Or attached via an alternative bond, such as a triazole bond (Li). In these compounds, Lii represents an internucleoside bond between the first region (A) and the second region (B). 5b shows a non-limiting example of a method for the synthesis of the compounds of the present invention. US stands for oligonucleotide synthesis support, which may be a solid support. X is a third region, such as a conjugate, targeting group, blocking group, and the like. In an optional previous step, X is added to the oligonucleotide synthesis support. Otherwise, a support with X already attached may be obtained (i). In the first stage, region B is synthesized (ii), followed by region A (iii), followed by cleavage of the oligomeric compound of the invention from the oligonucleotide synthesis support (iv). In an alternative method, the previous step involves providing an oligonucleotide synthesis support to which region X and linker group (Y) are attached (see FIG. 5a). In some embodiments, either X or Y (if present) is a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, such as a phosphonucleoside linking group, or a triazole linkage Attached to region B via an alternative bond. Non-limiting example of a method for synthesizing a compound of the present invention comprising a linker (Y) between the third region (X) and the second region (B). US stands for oligonucleotide synthesis support, which may be a solid support. X is a third region, such as a conjugate, targeting group, blocking group, and the like. In an optional previous step, Y is added to the oligonucleotide synthesis support. Otherwise, a support with Y already attached may be obtained (i). In the first stage, region B is synthesized (ii), followed by region A (iii), followed by cleavage of the oligomeric compound of the invention from the oligonucleotide synthesis support (iv). In some embodiments (as shown), region X can be added to the linker (Y) after the cleavage step (v). In some embodiments, Y is a region B via an alternative linkage such as a phosphonucleoside linkage group, such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, or a triazole linkage. To be attached to. Non-limiting examples of methods for synthesizing compounds of the present invention utilizing an activating group. In an optional previous step, an activating group is attached to the oligonucleotide synthesis support (i) or an oligonucleotide synthesis support having an activating group is obtained in another way. In step (ii), region B is synthesized, and then region A is synthesized (iii). The oligomer is then cleaved from the oligonucleotide synthesis support (iv). The intermediate oligomer (including the activating group) is then activated (vl) or (viii) to add a third region (X) (vi), optionally via a linker (Y) ( ix). In some embodiments, X (or Y, if present) is a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, such as a phosphonucleoside linkage group, or a triazole linkage, It is attached to region B via an alternative bond. A non-limiting example of a method for synthesizing a compound of the invention, wherein a bifunctional oligonucleotide synthesis support is used (i). In such a method, any oligonucleotide is synthesized in an initial series of steps (ii)-(iii) followed by attachment of a third region (optionally via linker group Y) The oligomeric compounds of the invention can then be cleaved (v). Alternatively, as shown in steps (vi)-(ix), a third region (optionally having a linker group (Y)) is attached to the oligonucleotide synthesis support (this is an optional pre-step -Alternatively, an oligonucleotide synthesis support having a third region (optionally with Y) is provided in another way and the oligonucleotide is then synthesized (vii-viii). The oligomeric compounds of the invention can then be cleaved (ix). In some embodiments, X (or Y, if present) is a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, such as a phosphonucleoside linkage group, or a triazole linkage, It is attached to region B via an alternative bond. In some embodiments, the US, in some embodiments, prior to the method (such as the previous step) is bi-directional (allowing independent synthesis of oligonucleotides and covalent attachment of the groups X, Y (or X and Y) ( It may include the step of adding a (difunctional) group to the support (as indicated)-this may be achieved, for example, using triazole or nucleoside groups. The bi-directional (bifunctional) group to which the oligomer is attached can then be cleaved from the support. Non-limiting example of a method for synthesizing a compound of the present invention: In the initial stage, a first region (A) is synthesized (ii) followed by region B. In some embodiments, a third region is then attached to region B (iii), optionally via a phosphate nucleoside bond (or, for example, a triazole bond). The oligomeric compounds of the invention can then be cleaved (iv). When a linker (Y) is used, in some embodiments, steps (v)-(viii) can be followed: After synthesis of region B, a linker group (Y) is added, followed by (Y In a subsequent step, region X is added (vi). The oligomeric compounds of the invention can then be cleaved (vii). In some embodiments, X (or Y, if present) is a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, such as a phosphonucleoside linkage group, or a triazole linkage, It is attached to region B via an alternative bond. Non-limiting example of a method for synthesizing a compound of the invention: In this method, an activating group is used: Steps (i) to (iii) are as in FIG. However, after oligonucleotide synthesis (step iii), an activating group (or reactive group) is added to region B, optionally via a phosphate nucleoside linkage. The oligonucleotide is then cleaved from the support (v). The activating group can then be activated to yield a reactive group, after which a third region (X), e.g., a conjugate, blocking group, or targeting group, can be activated by a reactive group (this can be an activated activating group). Or may be a reactive group) to give an oligomer (vi). As shown in (vii) to (viii), after cleavage, a linker group (Y) is added (vii), and then attached to (Y) or, in a subsequent step, region X is added. To yield oligomers (viii). In the alternative, all of steps (ii)-(viii) may be performed on the oligonucleotide synthesis support, and in such cases the final step of cleaving the oligomer from the support may be performed. It should be recognized that it is good. In some embodiments, the reactive or activating group is an alternative linkage, such as a phosphodiester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate, a phosphonucleoside linkage group, or a triazole linkage. Is attached to region B via Silencing of apo B mRNA by cholesterol conjugates in vivo. Mice were injected with a single dose of 1 mg / kg unconjugated LNA-antisense oligonucleotide (# 3833), or equimolar amounts of LNA antisense oligonucleotide conjugated to cholesterol with various linkers ( Table 3), sacrificed on days 1, 3, 7, and 10 after dosing. RNA was isolated from liver and kidney and subjected to apo B specific RT-qPCR. A. Quantification of apo B mRNA from liver samples normalized to GAPDH and presented as the average percentage of equivalent saline control. B. Quantification of apo B mRNA from kidney samples normalized to GAPDH and presented as the average percentage of equivalent saline control. Cholesterol C6 conjugates that can be used as X—Y— in the compounds of the present invention, as well as specific compounds used in the Examples, including specific compounds of the present invention. Cholesterol C6 conjugates that can be used as XY- in the compounds of the present invention, as well as specific compounds used in the Examples, including specific compounds of the present invention, are continued in Figure 12-1. Cholesterol C6 conjugates that can be used as XY- in the compounds of the present invention, as well as specific compounds used in the Examples, including specific compounds of the present invention, are continued from FIG. 12-2. Cholesterol C6 conjugates that can be used as XY- in the compounds of the present invention, as well as specific compounds used in the Examples, including the specific compounds of the present invention, are continued in Figure 12-3. Cholesterol C6 conjugates that can be used as X—Y— in the compounds of the present invention, as well as specific compounds used in the Examples, including specific compounds of the present invention, are continued in FIG. 12-4. Examples of cholesterol, trivalent GalNac, FAM, folic acid, monovalent GalNac, and tocopherol conjugates (eg, the compound of FIG. 12) used in the experiments. Examples of cholesterol, trivalent GalNac, FAM, folic acid, monovalent GalNac, and tocopherol conjugates (eg, the compound of FIG. 12) used in the experiment are continued from FIG. 13-1. Silencing of apo B mRNA by cholesterol conjugates in vivo. Mice were injected with a single dose of 1 mg / kg unconjugated LNA-antisense oligonucleotide (# 3833), or equimolar amounts of LNA antisense oligonucleotide conjugated to cholesterol with various linkers ( Table 3), sacrificed on days 1, 3, 7, 10, 13 and 16 after dosing. RNA was isolated from liver and kidney and subjected to apo B specific RT-qPCR. A. Quantification of apo B mRNA from liver samples normalized to GAPDH and presented as the average percentage of equivalent saline control. B. Quantification of apo B mRNA from kidney samples normalized to GAPDH and presented as the average percentage of equivalent saline control. Specific LNA oligonucleotide content in liver and kidney in vivo. Mice were injected with a single dose of 1 mg / kg unconjugated LNA-antisense oligonucleotide (# 1), or equimolar amounts of LNA antisense oligonucleotide conjugated to cholesterol with various linkers ( Table 4), sacrificed on days 1, 3, 7, 10, 13 and 16 after dosing. LNA oligonucleotide content was determined using a sandwich ELISA method based on LNA. Silencing of PCSK9 mRNA by cholesterol conjugates in vivo. Mice were injected with a single dose of 10 mg / kg unconjugated LNA-antisense oligonucleotide (# 7) or equimolar amounts of LNA antisense oligonucleotide conjugated to cholesterol with various linkers ( Table 5), sacrificed on days 1, 3, 7 and 10 after dosing. RNA was isolated from liver and kidney and subjected to PCSK9 specific RT-qPCR. A. Quantification of PCSK9 mRNA from liver samples normalized to BACT and presented as the average percentage of equivalent saline control. B. Quantification of PCSK9 mRNA from kidney samples normalized to BACT and presented as the average percentage of equivalent saline control. Examples of tri-GalNac conjugates that can be used. Conjugates 1 to 4 illustrate four suitable GalNac conjugate moieties, and conjugates 1a to 4a are the same conjugate, where the conjugate is an oligomer (region A or a biocleavable linker, such as a region Shown with an additional linker moiety (Y) used to link to B). The wavy line represents the covalent bond to the oligomer. Examples of cholesterol and tocopherol conjugate moieties are also shown (5a and 6a). The wavy line represents the covalent bond to the oligomer. An example of a tri-GalNac conjugate that can be used and is a continuation of FIG. 17-1. Conjugates 1 to 4 illustrate four suitable GalNac conjugate moieties, and conjugates 1a to 4a are the same conjugate, with the conjugate being an oligomer (region A or biocleavable linker, such as region B). Those with additional linker moieties (Y) used to link are shown. The wavy line represents the covalent bond to the oligomer. Examples of cholesterol and tocopherol conjugate moieties are also shown (5a and 6a). The wavy line represents the covalent bond to the oligomer. Fig. 17-2 is an example of a tri-GalNac conjugate that can be used. Conjugates 1 to 4 illustrate four suitable GalNac conjugate moieties, and conjugates 1a to 4a are the same conjugate, with the conjugate being an oligomer (region A or biocleavable linker, such as region B). Those with additional linker moieties (Y) used to link are shown. The wavy line represents the covalent bond to the oligomer. Examples of cholesterol and tocopherol conjugate moieties are also shown (5a and 6a). The wavy line represents the covalent bond to the oligomer. An example of a tri-GalNac conjugate that can be used and is a continuation of FIG. 17-3. Conjugates 1 to 4 illustrate four suitable GalNac conjugate moieties, and conjugates 1a to 4a are the same conjugate, with the conjugate being an oligomer (region A or biocleavable linker, such as region B). Those with additional linker moieties (Y) used to link are shown. The wavy line represents the covalent bond to the oligomer. Examples of cholesterol and tocopherol conjugate moieties are also shown (5a and 6a). The wavy line represents the covalent bond to the oligomer. Example 7a: Serum protein level of FVII Example 7a: FVII mRNA levels in the liver on day 4 Example 7a: Oligonucleotide content in liver and kidney on day 4 Example 7b-Serum protein level of FVII FVII mRNA levels in the liver on day 24 Oligonucleotide content in liver and kidney on day 4 In vivo silencing of apo B mRNA with various conjugates and PO-linkers. Treat mice with ASO 1 mg / kg with various conjugates, either with no biocleavable linker, with dithio-linker (SS), or with DNA / PO-linker (PO) did. RNA was isolated from liver (A) and kidney samples (B) and analyzed for apo B mRNA knockdown. Data are shown relative to saline (= 1). In vitro silencing of target X mRNA by loop LNA ASO with PO-linker. Neuro 2a cells were treated with loop LNA ASO with or without PO-linker, respectively. Six days later, gymnosis mRNA was extracted and analyzed for target X mRNA knockdown. mRNA expression is shown as a percentage of mock-treated samples.

DESCRIPTION OF INVENTION d relates to an oligomeric compound, such as an antisense oligonucleotide, covalently linked to a blocking group.

Oligomer The present invention utilizes oligomeric compounds (also referred to herein as oligomers) for use in modulating, eg inhibiting, target nucleic acids in cells. The oligomer may have a length of 8-35 contiguous nucleotides, comprising a first region of 7-25 contiguous nucleotides, and a second region of 1-10 contiguous nucleotides; For example, the internucleoside linkage between the first region and the second region is linked to the first (or only) DNA or RNA nucleoside of the second region by a phosphodiester, or the region B is at least 1 DNA nucleosides or RNA nucleosides linked by phosphodiesters.

  The second region can include, in some embodiments, additional DNA or RNA nucleosides that can be linked by phosphodiester. The second region is further covalently linked to a third region, which can be, for example, a conjugate, targeting group, reactive group, and / or blocking group.

  In some aspects, the invention provides a second region that is a labile region that links a first region, eg, an antisense oligonucleotide, to a conjugate group or functional group, eg, a targeting group or blocking group. Based on offer. An unstable region is a nucleoside linked by at least one phosphodiester, such as a DNA nucleoside or an RNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, Includes nucleosides linked by 9 or 10 phosphodiesters, such as DNA or RNA. In some embodiments, the oligomeric compound comprises a cleavable (labile) linker. In this regard, the cleavable linker is preferably present in region B (or in some embodiments, between region A and region B).

  The term “oligomer” in the context of the present invention refers to a molecule (ie, an oligonucleotide) formed by the covalent attachment of two or more nucleotides. Herein, a single nucleotide (unit) may also be referred to as a monomer or unit. In some embodiments, the terms “nucleoside”, “nucleotide”, “unit”, and “monomer” are used interchangeably. When referring to a nucleotide or monomer sequence, it will be appreciated that what is referred to is a sequence of bases such as A, T, G, C, or U.

  The oligomer is 8-25 nucleotides long, for example, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides , 19 nucleotide length, 20 nucleotide length, 21 nucleotide length, 22 nucleotide length, 23 nucleotide length, 24 nucleotide length, 25 nucleotide length, eg, 10-20 nucleotides in length or comprising a continuous nucleotide sequence.

  In various embodiments, the compounds of the invention do not comprise RNA (units). In some embodiments, a compound according to the invention, a first region, or a combination of a first region and a second region (eg, as a single continuous sequence) is a linear molecule or linear It is synthesized as a molecule. Thus, the oligomer can be a single-stranded molecule. In some embodiments, the oligomer comprises a short region that is complementary to an equivalent region within the same oligomer, eg, a region of at least 3 contiguous nucleotides, 4 contiguous nucleotides, or 5 contiguous nucleotides (ie, a duplex) Absent. The oligomer may not be (essentially) double stranded in some embodiments. In some embodiments, the oligomer is not essentially double stranded, eg, not siRNA.

  In some embodiments, the oligomer is a short region that is complementary to a region within the same first region, eg, a region of at least 3 contiguous nucleotides, 4 contiguous nucleotides, or 5 contiguous nucleotides (ie, an intraregion duplex). The first region that does not include may be included. In this regard, the first oligomer may not form a hybridization with a non-covalently linked complementary strand in some embodiments, eg, does not form part of an siRNA.

  For example, in some embodiments, the oligomeric compound comprises a complementary region, for example when the first region forms part of an siRNA, or when the third region comprises an aptamer or blocking oligonucleotide, for example. The oligomeric compounds of the present invention may in some embodiments comprise a region of double stranded nucleic acid. In such embodiments, a region of a double-stranded nucleic acid that forms a duplex, for example, at least 3 nucleotides long, such as at least 4 nucleotides long, such as at least 5 nucleotides long, such as at least 6 nucleotides long, is a third. In a region, or between a third region and, for example, a first region, or in some embodiments, in a second region, or a region spanning the first region and the second region (e.g., When the third region includes an oligonucleotide blocking region).

  In some embodiments, the oligomeric compound is not in duplex form with (substantially) complementary oligonucleotides, eg, is not siRNA.

  In some embodiments, the oligomeric compound is an LNA oligomer, eg, an LNA antisense oligomer, comprising an antisense oligomer, region B as defined herein, and a carbohydrate conjugate (which may be referred to as region C). (Which may be referred to herein as region A). LNA antisense oligomers can be 7-30 nucleosides long, eg, 8-26 nucleosides long, and include at least one LNA unit (nucleoside). In some embodiments, the carbohydrate moiety is not a linear carbohydrate polymer.

  In some embodiments, the oligomeric compound comprises an antisense oligomer, region B as defined herein, and an asialoglycoprotein receptor targeting moiety conjugate moiety, such as a GalNAc moiety (which may be referred to as region C). LNA oligomers, including, for example, LNA antisense oligomers (which may be referred to herein as region A). The carbohydrate moiety may be multivalent, eg, divalent, trivalent, tetravalent, or four identical or non-identical carbohydrate moieties optionally via a linker (such as region Y). And may be covalently bonded to the oligomer.

First region In some embodiments, the first region can comprise a nucleic acid-based oligomer, eg, an antisense oligonucleotide. In some embodiments, the first region comprises or consists of an oligonucleotide, eg, an antisense oligonucleotide, linked by a phosphorothioate 7-25 nucleotides in length. The first region can include at least one modified nucleoside (nucleoside analog), such as at least one bicyclic nucleoside (eg, LNA) or a 2′-substituted nucleoside. In some embodiments, some or all of the first region nucleosides may be modified nucleosides, also referred to herein as nucleoside analogs. In some embodiments, the modified nucleoside is sugar modified (eg, includes a sugar or sugar substitute moiety other than ribose or deoxyribose).

  In some embodiments, the first region is an antisense oligomer (antisense oligonucleotide) comprising a sequence complementary to the nucleic acid target, eg, a single stranded oligomer.

  In some embodiments, the first region comprises or is a gapmer. In some embodiments, the first region comprises or is a mixmer. In some embodiments, the first region comprises or is a totalmer.

  In some embodiments, the first region has at least 1, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 At least 23, at least 24, or 25 nucleoside analogs. In some embodiments, the nucleoside analog is (optionally) selected from the group consisting of a bicyclic nucleoside analog (eg, LNA) and / or a 2 ′ substituted nucleoside analog, eg, (optionally independent) 2) -O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, 2'-AP, 2'-FANA, 2 '-(3-hydroxy) propyl, and 2 '-Fluoro-DNA units, and / or other (optional) sugar-modified nucleoside analogs such as morpholinos, peptide nucleic acids (PNA), CeNA, unlinked nucleic acids (UNA), hexitol nucleic acids (HNA) , Selected from the group consisting of bicyclo-HNA (see eg WO2009 / 100320). In some embodiments, the nucleoside analog increases the affinity of the first region for its target nucleic acid (or complementary DNA or RNA sequence). Various nucleoside analogs are described by Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293, incorporated herein by reference. -213.

  In some embodiments, the oligomer, eg, its first region, eg, gapmer, mixmer, or totalmer, comprises at least one bicyclic nucleotide analogue, eg, LNA. In some embodiments, the first region comprises at least one bicyclic nucleoside analog (eg, LNA) and / or a 2 ′ substituted nucleoside analog. In some embodiments, the nucleoside analogs present in the first region all contain the same sugar modification. In some embodiments, the at least one nucleoside analog present in the first region is a bicyclic nucleoside analog, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, for example, all nucleoside analogs (Or in the totalmer, all nucleosides) are bicyclic nucleoside analogs such as LNA, eg, β-DX-LNA or α-LX-LNA (where X is oxy, amino, or thio) Or other LNAs disclosed herein, including, but not limited to, (R / S) cET, cMOE, or 5′-Me-LNA. In some embodiments, the oligomer or first region thereof comprises DNA and sugar-modified nucleoside analogs, such as bicyclic nucleoside analogs and / or 2 ′ substituted nucleoside analogs. In some embodiments, the oligomer or first region thereof comprises DNA and LNA nucleoside analogs.

  WO05013901, WO07 / 027775, WO07027894 refer to fully 2 ′ substituted oligomers such as fully 2′-O-MOE. In some embodiments, the first region of the oligomer may comprise a 2 ′ substituted nucleoside. WO07 / 027775 also mentions MOE, LNA, DNA mixmers for use to target microRNAs.

  In some embodiments, the first region, or a combination of the first region and the second region, does not comprise a region of 4 or more than 5 consecutive DNA units. Such a first region may be (essentially) unable to recruit RNAseH.

  The first region is covalently linked to the second region via, for example, a 5 ′ or 3 ′ terminal internucleoside linkage, eg, a phosphodiester linkage. Thus, the phosphodiester linkage is between the most 5 ′ nucleoside of region A and the most 3 ′ nucleoside of region B and / or the most 3 ′ nucleoside of region A and the most of region B. It can be located between the nucleoside in the 5 '. In this regard, in some embodiments, two regions B covalently joined to region A are present, one at the 5 ′ end of region A and one at the 3 ′ end of region A. May be. The two regions B may be the same or different and optionally independently linked to the same or different third region via a linker (Y). .

  In some embodiments, some or all of the nucleosides of the first region are modified nucleosides, also referred to herein as nucleoside analogs, such as sugar-modified nucleoside analogs, such as bicyclic nucleoside analogs (e.g., , LNA) and / or 2 ′ substituted nucleoside analogs. In some embodiments, the nucleoside analogs present in the first region all comprise the same sugar modification, e.g., all bicyclic nucleoside analogs such as LNA, e.g., β-DX-LNA or Disclosed herein, including α-LX-LNA (where X is oxy, amino, or thio) or (R / S) cET, cMOE, or 5′-Me-LNA, including but not limited to Other LNAs.

  In some embodiments, the internucleoside linkage of the first region comprises at least one internucleoside linkage other than a phosphodiester, such as at least one of the internucleoside linkages in region A, such as at least 50%, such as At least 75%, such as at least 90%, such as 100%, are other than phosphodiester. In some embodiments, internucleoside linkages other than phosphodiester are sulfur-containing internucleoside linkages, such as phosphorothioates, phosphodithioates, and boranophosphates, such as phosphorothioates.

Second area (Area B)
The second region may comprise or consist of at least one DNA or RNA nucleoside linked to the first region via a phosphodiester linkage. In some aspects, the internucleoside linkage between the first region and the second region is considered part of region B.

  In some embodiments, the second region has at least 1-10 linked nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 Or comprises or consists of 10 linked DNA or RNA nucleotides. The region of DNA / RNA phosphodiester is considered important in providing a cleavable linker, but region B may also contain sugar-modified nucleoside analogs, such as those mentioned for the first region above. Is possible. However, in some embodiments, the nucleoside of region B is (optionally) selected from the group consisting of DNA and RNA. It will be appreciated that the nucleoside of region B may comprise a naturally occurring nucleobase or a non-naturally occurring nucleobase. Region B comprises a DNA or RNA nucleoside linked by at least one phosphodiester (which in some embodiments may be the first nucleoside adjacent to region A). Where region B includes other nucleosides, region B may also include other nucleoside linkages other than phosphodiesters, such as (optionally) phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate. However, in other embodiments, all internucleoside linkages within region B are phosphorothioate. In some embodiments, all nucleosides in region B (optionally independently) comprise either a 2′-OH ribose sugar (RNA) or a 2′-H sugar (ie, RNA or DNA).

  In some embodiments, the second region comprises at least 1-10 (eg, by phosphodiester) linked DNA or RNA nucleosides, such as 1, 2, 3, 4, 5, Contains or consists of 6, 7, 8, 9, or 10 linked DNA or RNA nucleotides (eg, by phosphodiester).

  In some embodiments, region B comprises no more than 3 or no more than 4 consecutive DNA or RNA nucleosides, eg, DNA nucleosides. Thus, region B may be short so as not to recruit RNAseH, and this aspect may be important when region B does not form part of a single contiguous nucleobase sequence that is complementary to the target. Shorter regions B, eg, 1-4 nt long regions B, may also be preferred in some embodiments because they are less likely to be sequence specific restriction enzyme targets. Thus, it is possible to vary the sensitivity of region B to endonuclease cleavage, thereby fine-tuning the rate of activation of the active oligomers in vivo or intracellularly. Where very rapid activation is required, optionally include longer region B and / or restriction enzyme recognition sites (eg, specific or differentially expressed in cells or tissues) Region B can be utilized.

  As illustrated in the examples, region B can include, for example, a phosphodiester linkage, and / or any suitable linker group, such as those provided herein, such as a phosphate nucleoside linkage (eg, phospho Diester, phosphorothioate, phosphorodithioate, boranophosphate, or methylphosphonate), or a linker group that may contain a triazole group, via a conjugate group, targeting group, reactive group, activating group, or blocking group (X) Can be conjugated. In some aspects, the linking group is the same as the linking group between region A and region B, and thus can be a phosphodiester linkage. In some aspects, the linking group is a phosphorothioate linkage.

  In some embodiments, the DNA or RNA nucleotides of the second region are independently selected from DNA and RNA nucleotides. In some embodiments, the DNA nucleotide or RNA nucleotide of the second region is a DNA nucleotide. In some embodiments, the DNA nucleotide or RNA nucleotide of the second region is an RNA nucleotide.

  With respect to the second region, the terms DNA nucleoside and RNA nucleoside may include naturally occurring bases or non-naturally occurring bases (also called base analogs or modified bases).

  It will be appreciated that in some embodiments, the second region may further comprise other nucleotides or nucleotide analogs. In some embodiments, the second region comprises only DNA nucleosides or RNA nucleosides. In some embodiments, when the second region comprises a plurality of nucleosides, the internucleoside linkage in the second region comprises a phosphodiester linkage. In some embodiments, when the second region comprises a plurality of nucleosides, all internucleoside linkages in the second region comprise phosphodiester linkages.

  In some embodiments, the at least two contiguous nucleosides of the second region are DNA nucleosides (eg, at least 3, or 4 or 5 contiguous DNA nucleotides). In some embodiments, the at least two contiguous nucleosides of the second region are RNA nucleosides (eg, at least 3, or 4 or 5 contiguous RNA nucleotides). In some embodiments, the at least two contiguous nucleosides of the second region are at least one DNA and at least one RNA nucleoside. The internucleoside linkage between region A and region B is a phosphodiester linkage. In some embodiments, when region B comprises a plurality of nucleosides, at least one additional internucleoside linkage, eg, 2 (or 3 or 4 or 5) nucleosides adjacent to region A The linking group between is a phosphodiester.

  One side of the second region (either 5 'or 3') is flanked by the first region, eg, an antisense oligonucleotide, and the other side (either 3 'or 5', respectively) Optionally (ie, between the second region and a moiety such as a conjugate / blocking group) via a linker group, a conjugate moiety or similar group (eg, blocking moiety / group, targeting moiety / group, or therapeutic Small molecule part) is adjacent.

In such embodiments, the oligonucleotides of the invention can be illustrated by the following formula:
5'-A-PO-B [Y] X-3 'or 3'-A-PO-B [Y] X-5'
Where A is region A, PO is a phosphodiester linkage, B is region B, Y is any linking group, X is a conjugate group, targeting group, blocking group. Or a reactive or activating group.

In some embodiments, region B is 3 ′ to 5 ′ or 5 ′ to 3 ′: (i) a phosphodiester linkage with the 5 ′ nucleoside of region A, (ii) a DNA nucleoside or RNA nucleoside, such as DNA Including a nucleoside, and (iii) an additional phosphodiester linkage.
5'-A-PO-B-PO-3 'or 3'-A-PO-B-PO-5'

  Further phosphodiester linkages link the nucleoside of region B to one or more additional nucleosides, eg, one or more DNA nucleosides or RNA nucleosides, or optionally via a linking group (Y), X ( A conjugate group, a targeting group, or a blocking group, or a reactive group or an activating group).

In some embodiments, region B is 3 ′ to 5 ′ or 5 ′ to 3 ′: (i) a phosphodiester linkage with 5 ′ nucleoside of region A, (ii) 2 to 10 phosphodiesters Concatenated DNA or RNA nucleosides, such as DNA nucleosides, and optionally (iii) further phosphodiester linkages:
5'-A- [PO-B] n- [Y] -X 3 'or 3'-A- [PO-B] n- [Y] -X 5'
5'-A- [PO-B] n-PO- [Y] -X 3 'or 3'-A- [PO-B] n-PO- [Y] -X 5'
In the formula, A represents the region A, [PO-B] n represents the region B, and n represents 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 Or PO and PO is an optional phosphodiester linking group between region B and X (or Y, if present).

In some embodiments, the present invention provides a compound according to (or including) one of the following formulas:
5 '[Area A] -PO- [Area B] 3'-YX
5 '[Area A] -PO- [Area B] -PO 3'-YX
5 '[Area A] -PO- [Area B] 3'-X
5 '[Area A] -PO- [Area B] -PO 3'-X
3 '[Area A] -PO- [Area B] 5'-YX
3 '[Area A] -PO- [Area B] -PO 5'-YX
3 '[Area A] -PO- [Area B] 5'-X
3 '[Area A] -PO- [Area B] -PO 5'-X

Region B includes or consists of, for example:
5 'DNA 3'
3 'DNA 5'
5 'DNA-PO-DNA-3'
3 'DNA-PO-DNA-5'
5 'DNA-PO-DNA-PO-DNA 3'
3 'DNA-PO-DNA-PO-DNA 5'
5 'DNA-PO-DNA-PO-DNA-PO-DNA 3'
3 'DNA-PO-DNA-PO-DNA-PO-DNA 5'
5 'DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 3'
3 'DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 5'

Sequence selection in the second region:
In some embodiments, region B does not form a complementary sequence when oligonucleotide regions A and B are aligned with complementary target sequences.

  In some embodiments, region B forms a complementary sequence when oligonucleotide regions A and B are aligned with complementary target sequences. In this regard, region A and region B may together form a single continuous sequence that is complementary to the target sequence.

  In some embodiments, the sequence of bases in region B is selected based on the predominant endonuclease cleavage enzyme present in the target tissue or cell or intracellular compartment to provide the optimal endonuclease cleavage site. In this regard, by isolating cell extracts from target and non-target tissues, preferential cleavage in the desired target cells (eg, liver / hepatocytes) compared to non-target cells (eg, kidney) Based on the activity, an endonuclease cleavage sequence for use in region B can be selected. In this regard, the efficacy of the compound for target downregulation can be optimized for the desired tissue / cell.

  In some embodiments, region B has the sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, where C is 5-methylcytosine And / or T may be replaced with U). In some embodiments, region B comprises the sequences AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG , TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA , CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, and GGG (C may be 5-methylcytosine Often and / or T may be replaced with U). In some embodiments, region B comprises the sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX , TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX, CCGX, CGAX , CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX (X, A, T, U, G , C, and analogs thereof, wherein C may be 5-methylcytosine and / or T may be replaced with U). When referring to (naturally occurring) nucleobases A, T, U, G, C, these function as equivalent natural nucleobases (eg, base paired with complementary nucleosides). It will be appreciated that may be substituted.

  In some embodiments, a compound of the invention comprises a plurality of conjugate groups (or a plurality of functional groups X—eg, conjugate groups, targeting groups, blocking groups, or activated groups, or reactive groups or Activating groups), for example 2 or 3 such groups may be included. In some embodiments, region B is covalently linked to at least one functional group, such as two or three functional groups, optionally via a [eg, non-nucleotide] linker group. In some embodiments, the first region may be covalently linked to two regions B (eg, one 5 ′ of the first region, one 3 ′) (eg, a nucleoside). Each region B (via an interlinkage, eg, via a phosphodiester linkage) can (optionally and independently) be selected from region B described herein. In this respect, one region B may have one or more functional groups, and the second region B may also have one or more functional groups, and each region B has a functional group. Can be independently selected from conjugates, targeting groups, blocking groups, or reactive / activating groups.

Poly-oligomer compound The present invention may comprise a first region (region A), a second region (region B), and a third region (region C), wherein the first region comprises at least one additional oligomeric compound ( A biocleavable linker that is covalently linked to region A ′) and wherein the first region (region A) and region A ′ can be, for example, similar to those of the second region disclosed herein (Region B ′), eg, DNA or RNA (eg, DNA) linked by at least one phosphodiester, eg, a DNA nucleoside linked by 2, 3, 4, or 5 phosphodiester Alternatively, polyoligomeric compounds are provided that are covalently linked through regions of RNA nucleosides (eg, DNA nucleosides). Regions B and B ′, in some embodiments, have the same structure, eg, the same number of DNA / RNA nucleoside and phosphodiester linkages and / or the same nucleobase sequence. In other embodiments, regions B and B ′ may be different. For example, such a poly-oligomer compound can be (5′-3 ′ or 3′-5 ′) conjugate-PO-ON-PO′-ON ′ (conjugate is region C, PO is region B PO ′ is the region B ′, ON 1 is the region A, and ON ′ is the region A ′).

  Region A ′, in some embodiments, is a plurality of additional oligomeric compounds (eg, two or three additional oligomeric compounds) linked in a row (or in parallel) via a biocleavable linker, for example, Conjugate-PO-ON-PO-ON'-PO ''-ON '' or conjugate-PO-ON- [PO-ON '] n (n can be, for example, 1, 2, or 3, It is understood that each ON 'may include the same or different and, if different, may have the same target or different targets) It should be.

Multiconjugate oligomeric compound In some embodiments, the oligomeric compound may be conjugated to multiple conjugate regions (region C), which may be the same or different. For example, an oligomeric compound of the invention may have the following structure: (5′-3 ′ or 3′-5 ′) ON-PO′-conjugate 1-PO ″ -conjugate 2 (conjugate Gate 1 and conjugate 2 are two conjugate groups, at least one or both of PO or PO '' is the same as in region B herein, and ON is in region A Is). Conjugate 1 and conjugate 2 may be the same or different. For example, in some embodiments, one of Conjugate 1 and Conjugate 2 is a carbohydrate or sterol conjugate, and the other is a lipophilic conjugate, such as 5′-3 ′ or 3′- 5 ′: ON-PO′-palmitoyl-PO ″ -cholesterol or ON-PO′-palmitoyl-PO ″ -GalNac.

  The carbohydrate conjugate moiety represented by GalNac in the above formula (eg when used as conjugate 1 or conjugate 2) is, for example, galactose, galactosamine, N-formyl-galactosamine, N acetylgalactosamine, N-propionyl- It can be selected from the group consisting of galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactose-amine. The lipophilic conjugate (eg, when used as conjugate 1 or conjugate 2 and represented in the above formula as palmitoyl) can be a hydrophobic group, eg, a C16-20 hydrophobic group, a sterol, cholesterol. Other carbohydrates and lipophilic groups that can be used are disclosed herein, for example.

Target In some embodiments, as a non-limiting example, the oligomer of the invention is a nucleic acid selected from the group consisting of mRNA, microRNA, lncRNA (long non-coding RNA), snRNA, snoRNA, and viral RNA ( That is, for use in target) regulation.

  Exemplary but non-limiting mRNA and microRNA targets include, for example:

Genes shown in cancer, such as Hif1-α, survivin, Bcl2, Mcl1, Her2, androgen receptor, β-catenin, human transforming growth factor TGF-β2, ras, TNF-α, c-RAF, HSP For example, Hsp27, eIF-4E (eg, ISIS-EIF4ER _x ), STAT3 (eg, ISIS-STAT3Rx), clusterin (eg, OGX-011), AurkB, AurkA, PBK, miR-155, miR-21, miR-10b, mir-34 (see WO2011088309), miR-199a, miR-182.

  MRNA of genes involved in inflammation, such as ICAM-1 (eg Alicoforsen), CD49d, VLA-4, osteopontin, miR-21 (psoriasis).

  Other medically relevant mRNA targets include CTGF (local fibrosis) and c-Raf-kinase (eye disease), miR-29 (cardiac fibrosis), factor XI (coagulation), factor VII (coagulation) ), MiR15, miR-159 (modeling after myocardial infarction), miR-138 (bone loss), mir-21 (see WO12148952), and mir214 (fibrosis) (see WO2012012716).

  Targets of metabolic diseases or disorders such as Apo-B (high LDL cholesterol, ACS), Apo CIII (high serum TG, diabetes), Apo (a) (cardiovascular disease), FGFR4 (obesity), GCCR (T2 diabetes) ), GCGR (T2 diabetes), PTP1B (T2 diabetes), DGAT2 (NASH), PCSK9 (hyperlipidemia and related disorders), MtGPAT (obesity and NAFLD), miR-122 (high cholesterol), miR-33 (metabolism) Syndrome, atherosclerosis), miR-208 (chronic heart failure), miR-499 (chronic heart failure), miR-378 (cardiovascular metabolic disease), mir-143 (vascular disease), miR-145 (vascular disease) , MiR-92 (peripheral artery disease), miR-375 (diabetes), miR-27b (diabetes), miR-34a (diabetes), miR-199a, miR-27a (heart disease, ischemia), miR-338 ( Diabetes mellitus).

  Metabolic diseases include, for example, metabolic syndrome, obesity, hyperlipidemia, HDL / LDL cholesterol imbalance, dyslipidemia such as familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin Resistant hypercholesterolemia, coronary artery disease (CAD), coronary heart disease (CHD), atherosclerosis, heart disease, diabetes (type I and / or type II), NASH, acute coronary syndrome (ACS) included.

  Viral diseases: miR-451 (erythrocytosis), miR-122 (HCV), HBV, HCV, BKV, etc. Severe rare diseases include SMN2 (spinal muscular atrophy), TTR (TTR amyloidosis), GHr (acromegaly), AAT (AATD-related liver disease), dystrophin (Duchenne muscular dystrophy).

  In some embodiments, the oligomers of the invention target nucleic acid expressed in the liver, eg, mRNA expressed in the liver, eg, PCSK9, Apo B, or MtGPAT. In some embodiments, the oligomer of the invention targets PCSK9 mRNA. In some embodiments, the oligomer of the invention targets apo B mRNA. In some embodiments, the oligomer of the invention targets a microRNA expressed in the liver, eg, miR-122.

  In some embodiments, the oligomers of the invention can down regulate (eg, reduce or eliminate) expression of a target (eg, target nucleic acid). In this regard, the oligomers of the present invention can affect target inhibition. In some embodiments, an oligomer of the invention binds to a target nucleic acid and is at least 10% or 20% expressed compared to a normal expression level (eg, an expression level in the absence of the oligomer or conjugate). Inhibition, more preferably affects at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% inhibition compared to normal expression levels. In some embodiments, such modulation is seen when using a compound of the invention at a concentration of 0.04 to 25 nM, such as 0.8 to 20 nM. In the same or different embodiments, inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. is there. Modulation of expression levels can be determined by measuring protein levels by methods such as Western blotting using appropriate antibodies against the target protein after SDS-PAGE, for example. Alternatively, modulation of expression levels can be determined by measuring the level of mRNA, eg, by Northern blotting or quantitative RT-PCR. When measured via mRNA levels, the level of down-regulation when using an appropriate dosage, such as a concentration of 0.04-25 nM, such as 0.8-20 nM, is typically in some embodiments typically The level will be 10-20% of the normal level in the absence of the compound, conjugate, or composition.

  Accordingly, the present invention reduces the expression of a target in a cell expressing the target comprising the step of administering to the cell an oligomer or conjugate according to the present invention in order to down regulate or inhibit the expression of the target in the cell. Methods of controlling or inhibiting are provided. Suitably the cell is a mammalian cell, eg a human cell. Administration can occur in vitro in some embodiments. Administration can occur in vivo in some embodiments. The compounds of the invention, eg oligomers and their conjugates, can be targeted to various targets, eg mRNA or microRNA or other nucleic acid targets expressed in the liver (see NCBI Genbank / Gene ID reference , Given as examples of sequences that can be targeted by the compounds of the invention, the Genbank / NCBI sequence is incorporated herein by reference).

Apo B
In some embodiments, the first region (or the first and second regions) is an Apo B mRNA target, such as Apo B-100 (NCBI Genbank ID NM_000384.2 GI: 105990531 which is incorporated herein by reference). ) To form a single contiguous nucleobase sequence that is complementary (ie, targeted to).

  The compounds of the invention that target apo B can be used in the treatment of acute coronary syndromes (see WO20100076248). Accordingly, the present invention provides an oligomer according to the present invention that targets apo B100 for use in the treatment of acute coronary syndromes. The present invention further provides a method for the treatment of acute coronary syndrome comprising the step of administering the oligomer of the present invention to a subject in need of treatment.

  The compounds of the invention that target apo B can be used in the treatment of atherosclerosis. Accordingly, the present invention provides an oligomer according to the present invention that targets apo B100 for use in the treatment of atherosclerosis. The invention further provides a method of treating atherosclerosis comprising administering an oligomer of the invention to a subject in need of treatment. The compounds of the invention that target apo B can be used in the treatment of hypercholesterolemia or hyperlipidemia. Thus, the present invention provides an oligomer according to the present invention that targets apo B100 for use in the treatment of hypercholesterolemia or hyperlipidemia. The present invention further provides a method for the treatment of hypercholesterolemia or hyperlipidemia comprising the step of administering the oligomer of the present invention to a subject in need of treatment.

  The invention relates to the inhibition of apo B in cells expressing apo B, comprising the step of administering to the cell an oligomer or conjugate or pharmaceutical composition according to the invention to inhibit apo B in the cell. In vivo or in vitro methods are provided.

配列中、大文字は、β-D-オキシLNA単位（ヌクレオシド）であり、小文字は、DNA単位であり、下付きのsは、ホスホロチオエート連結であり、大文字のCの前の上付きのmは、全てのLNAシトシンが5-メチルシトシンであることを示す。アポBを標的とする本発明の化合物は、本明細書に開示される、オリゴマーを肝臓へ標的指向化するコンジュゲート、例えば、炭水化物または親油性コンジュゲート、例えば、GalNacコンジュゲートまたはステロールコンジュゲート（例えば、コレステロールもしくはトコフェロール）に、コンジュゲートされていてもよい。コンジュゲートは、例えば、（適宜領域Bを介して）オリゴマー化合物の5'末端または3'末端に在り得る。アポBを標的とするその他のオリゴマーは、WO03/011887、WO04/044181、WO2006/020676、WO2007/131238、WO2007/031081、およびWO2010142805に開示されている。 Examples of LNA oligomers that can be used as the first region in the oligomer / conjugate of the invention include, for example, those disclosed in WO2007 / 031081, WO2008 / 113830, WO2007131238, and WO2010142805, which are incorporated herein by reference. included. Specific preferred compounds include the following:

In the sequence, uppercase letters are β-D-oxy LNA units (nucleosides), lowercase letters are DNA units, subscript s is phosphorothioate linkage, and superscript m before uppercase C is All LNA cytosines are 5-methylcytosine. Compounds of the invention that target apo B are disclosed herein in conjugates that target oligomers to the liver, such as carbohydrate or lipophilic conjugates, such as GalNac conjugates or sterol conjugates (disclosed herein) For example, it may be conjugated to cholesterol or tocopherol). The conjugate can be, for example, at the 5 ′ end or 3 ′ end of the oligomeric compound (as appropriate through region B). Other oligomers that target apo B are disclosed in WO03 / 011887, WO04 / 044181, WO2006 / 020676, WO2007 / 131238, WO2007 / 031081, and WO2010142805.

PCSK9
In some embodiments, the first region (or first region and second region) is a PCSK9 mRNA target, eg, human PCSK9 mRNA (NCBI Genbank ID NM — 174936.3 GI: 299523249, incorporated herein by reference). A single contiguous nucleobase sequence is formed that is complementary to (ie, targets) the corresponding region.

  The present invention is for use as a medicament, e.g. of hypercholesterolemia or related disorders, e.g. atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, e.g. of PCSK9 Gain-of-function mutations, HDL / LDL cholesterol imbalance, dyslipidemia such as familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin resistant hypercholesterolemia, coronary artery disease (CAD), and Oligomers according to the invention targeting PCSK9 are provided for the treatment of disorders selected from the group consisting of coronary heart disease (CHD).

  The present invention relates to hypercholesterolemia or related disorders such as atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, eg, gain-of-function mutations in PCSK9, HDL / LDL cholesterol Group consisting of imbalance, dyslipidemia, eg familial hyperlipidemia (FCHL), acquired hyperlipidemia, statin resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD) There is provided the use of an oligomer of the invention targeting PCSK9 for the manufacture of a medicament for the treatment of a more selected disorder.

  The present invention comprises administering an effective amount of an oligomer according to the present invention targeting PCSK9 to a patient suffering from or likely to suffer from hypercholesterolemia or a related disorder. Disorders such as atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, eg, gain-of-function mutations in PCSK9, HDL / LDL cholesterol imbalance, dyslipidemia, eg, familial Provides a method for treating a disorder selected from the group consisting of hyperlipidemia (FCHL), acquired hyperlipidemia, statin resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD) To do.

  The present invention provides an in vivo or in vitro method for inhibiting PCSK9 in cells expressing PCSK9, comprising administering to the cell an oligomer according to the present invention that targets PCSK9 to inhibit PCSK9 in the cell. provide.

配列中、大文字は、β-D-オキシLNA単位（ヌクレオシド）であり、小文字は、DNA単位であり、下付きのsは、ホスホロチオエート結合であり、大文字のCの前の上付きのmは、全てのLNAシトシンが5-メチルシトシンであることを示す。PCSK9を標的とする本発明の化合物は、本明細書に開示される、オリゴマーを肝臓へ標的指向化するコンジュゲート、例えば、炭水化物または親油性コンジュゲート、例えば、GalNacコンジュゲートまたはステロールコンジュゲート（例えば、コレステロールもしくはトコフェロール）へコンジュゲートされていてもよい。コンジュゲートは、例えば、（適宜領域Bを介して）オリゴマー化合物の5'末端または3'末端に在り得る。PCSK9を標的とするその他のオリゴマーは、SEQ ID NO 36〜52として開示され、他は、参照によって本明細書に組み入れられるWO2008/043753、WO2011/009697、WO08/066776、WO07/090071、WO07/146511、WO0/143315、WO09/148605、WO11/123621、およびWO11133871に開示されている。 The following are oligomers that target human PCSK9 mRNA and can be used as region A in the compounds of the present invention.

In the sequence, uppercase letters are β-D-oxy LNA units (nucleosides), lowercase letters are DNA units, subscript s is a phosphorothioate bond, and superscript m before uppercase C is All LNA cytosines are 5-methylcytosine. The compounds of the invention that target PCSK9 are disclosed herein for conjugates that target oligomers to the liver, such as carbohydrate or lipophilic conjugates, such as GalNac conjugates or sterol conjugates (eg, , Cholesterol or tocopherol). The conjugate can be, for example, at the 5 ′ end or 3 ′ end of the oligomeric compound (as appropriate through region B). Other oligomers targeting PCSK9 are disclosed as SEQ ID NO 36-52, others are incorporated herein by reference, WO2008 / 043753, WO2011 / 009697, WO08 / 066776, WO07 / 090071, WO07 / 146511. , WO0 / 143315, WO09 / 148605, WO11 / 123621, and WO11133871.

の対応する領域に相補的である（即ち、それを標的とする）単一の連続核酸塩基配列を形成する。miR-122は、HCV感染において示されており、感染の維持のために必要とされる不可欠の宿主因子である。従って、miR-122の阻害剤は、C型肝炎感染の処置において使用され得る。 miR-122
In some embodiments, the first region (or first region and second region) is a microRNA-122, eg, miR-122a, eg, has-miR-122 sequence (miRBase release 20: MI0000442), eg,

Form a single contiguous nucleobase sequence that is complementary to (ie, targets) the corresponding region of. miR-122 has been shown in HCV infection and is an essential host factor required for the maintenance of infection. Thus, inhibitors of miR-122 can be used in the treatment of hepatitis C infection.

  The compounds of the invention that target miR-122 can be used in the treatment of HCV infection. Accordingly, the present invention provides an oligomer according to the present invention that targets miR-122 for use in the treatment of HCV infection. The present invention further provides a method for the treatment of HCV infection comprising the step of administering the oligomer of the present invention to a subject in need of treatment.

  The present invention provides the use of an oligomer of the invention targeting miR-122 for the manufacture of a medicament for the treatment of HCV infection.

  The present invention provides a method of treating HCV infection comprising administering to a patient suffering from HCV infection an effective amount of an oligomer according to the present invention targeting miR-122.

  The present invention relates to a cell expressing miR-122, for example an HCV infected cell, comprising administering to the cell an oligomer or conjugate or pharmaceutical composition according to the present invention to inhibit miR-122 in the cell. Alternatively, an in vivo or in vitro method for inhibition of miR-122 in HCV replicon expressing cells is provided.

  miR-122 has also been shown in cholesterol metabolism and it has been suggested that inhibition of miR-122 can be used for treatments that lower plasma cholesterol levels (Esau, Cell Metab. 2006 Feb; 3 (2): 87-98).

  Thus, inhibitors of miR-122 are metabolic diseases (related disorders) in treatment to lower plasma cholesterol levels or associated with elevated cholesterol levels, such as atherosclerosis, hyperlipidemia, high cholesterol Can be used in the treatment of an indication selected from the group consisting of dysemia, familial hypercholesterolemia, dyslipidemia, coronary artery disease (CAD), and coronary heart disease (CHD). The compounds of the invention that target miR-122 can be used in the treatment of elevated cholesterol levels or related disorders. Accordingly, the present invention provides an oligomer according to the present invention that targets miR-122 for use in the treatment of elevated cholesterol levels or related disorders. The invention further provides a method for the treatment of elevated cholesterol levels or related disorders comprising the step of administering an oligomer of the invention to a subject in need of treatment.

  The present invention provides the use of an oligomer of the invention targeting miR-122 for the manufacture of a medicament for the treatment of elevated cholesterol levels or related disorders.

  The present invention provides a method of treating elevated cholesterol levels or related disorders comprising administering to a patient suffering from a disorder an effective amount of an oligomer according to the present invention that targets miR-122.

  The present invention relates to a cell expressing miR-122, for example an HCV infected cell, comprising administering to the cell an oligomer or conjugate or pharmaceutical composition according to the present invention to inhibit miR-122 in the cell. Alternatively, an in vivo or in vitro method for inhibition of miR-122 in HCV replicon expressing cells is provided.

  Oligomers targeting miR-122 are disclosed in WO2007 / 112754, WO2007 / 112753, WO2009 / 043353 and are mixedmers such as SPC3649 (also referred to as miravirsen; see below) or small LNAs such as WO2009. / 043353 (for example, 5'-ACACTCC-3 ', 5'-CACACTCC-3', 5'-TCACACTCC-3 ', in the sequence, capital letters are β-D_oxyLNA, complete phosphorothioate And LNA C is 5-methylcytosine). In some embodiments, the oligomer targeting miR-122 is 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides ( Or 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the oligomer targeting miR-122 comprises a sequence that is completely complementary to miR-122 as measured over the entire length of the oligomer, preferably the sequence 5′-CACACTCC-3 'including. In some embodiments, the microRNA, eg, an oligomer that targets miR-122, is complementary to the corresponding region of the microRNA throughout the length of the oligomer, and in some embodiments, the oligomer 3 ′ The nucleoside is complementary to the first, second, third, or fourth 5 ′ nucleotide of a microRNA, eg, miR-122, eg, the second 5 ′ nucleotide of a microRNA, eg, miR-122 (Ie align with them).

本発明（領域A）に関して使用され得る、その他のmiR-122を標的とする化合物は、WO2007/027894、WO2007/027775に開示されている。 The following are oligomers that target has-miR-122 (human miR-122) and can be used as region A in the compounds of the present invention.

Other miR-122 targeted compounds that can be used in connection with the present invention (region A) are disclosed in WO2007 / 027894, WO2007 / 027775.

  MtGPAT: (NCBI gene ID 57678-chromosome: 10; NC_000010.10 (113907971..113975153, complementary strand) Mitochondrial glycerol-3-phosphate acyltransferase 1 (EC 2.3.1.15, also known as GPAT1, mtGPAT1, GPAM, mtGPAM) ) Plays a major role in triglyceride formation in the liver, high levels of mtGPAT1 activity result in fatty liver (hepatic steatosis), and lack of mtGPAT1 results in low levels of liver triglycerides and stimulated fatty acid oxidation (See WO2010 / 000656, which discloses oligomers targeting mtGPAT.) The compounds of the invention targeting MtGPAT are overweight, obese, fatty liver, hepatic steatosis, non-alcoholic fatty Treat conditions such as liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, diabetes, eg non-insulin dependent diabetes mellitus (NIDDM) Can be used for.

  Factor VII (NCBI gene ID 2155, NCBI J02933.1 GI: 180333, or EU557239.1 GI: 182257998). The oligomers or conjugates of the present invention can target Factor VII, thereby inhibiting the production of Factor VII, a critical component of the tissue factor coagulation pathway. Compounds of the invention that target factor VII are (typically without causing bleeding) for the treatment or prevention of thrombotic diseases such as heart attacks, strokes, and blood clots, or inflammatory conditions Can be used. WO2013 / 119979 and WO2012 / 174154, incorporated herein by reference, disclose oligonucleotide compounds targeting FVII that can be incorporated into the conjugates of the invention.

  Factor XI (NCBI Genbank BC122863.1 GI: 114108211)-Factor XI, a coagulation factor produced in the liver. High levels of factor XI are associated with heart attacks, strokes, and blood clots. WO2013 / 070771, incorporated herein by reference, discloses oligonucleotide compounds targeting XI that can be incorporated into the conjugates of the invention. The compounds of the invention that target Factor XI can be used for the treatment or prevention of thrombotic diseases such as heart attacks, strokes, and blood clots, or inflammatory conditions such as arthritis and colitis.

  Apo CIII (NCBI Genbank BC027977.1 GI: 20379764) A protein that regulates blood triglyceride metabolism. High levels of apo C-III are associated with inflammation, high triglycerides, atherosclerosis, and metabolic syndrome. The compounds of the invention that target apo CIII are used to reduce serum triglyceride levels as a single agent or in combination with other triglyceride lowering agents, or for example, familial chylomicronemia syndrome and severe hypertriglycerides Can be used in the treatment of WO11085271, incorporated herein by reference, discloses oligonucleotide compounds targeting apo CIII that can be incorporated into the conjugates of the invention.

  Apo (a) (NCBI Genbank NM_005577.2 GI: 116292749) presents a direct approach to inhibit the production of apo (a) in the liver and reduce Lp (a), an independent risk factor for heart disease Designed for. High levels of Lp (a) are associated with increased risk of atherosclerosis, coronary heart disease, heart attacks, and stroke. Lp (a) promotes premature plaque development or atherosclerosis in arteries. The compounds of the invention that target apo (a) can be used, for example, in the treatment of atherosclerosis and coronary heart disease. WO05000201 and WO03014307, incorporated herein by reference, disclose oligonucleotide compounds targeting apolipoprotein (a) that can be incorporated into the conjugates of the invention.

  Hepatitis B (HBV) (eg NCBI D23684.1 GI: 560092; D23683.1 GI: 560087; D23682.1 GI: 560082; D23681.1 GI: 560077; D23680.1, all incorporated herein by reference) GI: 560072; D23679.1 GI: 560067; D23678.1 GI: 560062; D23677.1 GI: 560057)

  Oligomers that target HBV are well known in the art. For example, see WO96 / 03152, WO97 / 03211, WO2011 / 052911, WO2012 / 145674, WO2012 / 145697, WO2013 / 003520, and WO2013 / 159109.

  The compounds of the invention that target HBV can be used in the treatment of HBV infection. Accordingly, the present invention provides an oligomer according to the present invention that targets HBV for use in the treatment of HBV. The present invention further provides a method of treatment of HBV infection comprising administering an oligomer of the invention to a subject in need of treatment.

  The present invention provides oligomers or conjugates of the invention that target hepatitis B (HBV) for use as a medicament, eg, for the treatment of hepatitis B infection or related disorders.

である。さらなる化合物は、参照によって本明細書に組み入れられる、WO2011/47312の表1、ならびにWO2011/052911、WO2012/145674、WO2012/145697、WO2013/003520、およびWO2013/159109に開示されている。 The invention provides the use of an oligomer or conjugate or pharmaceutical composition according to the invention targeting hepatitis B (HBV) for the manufacture of a medicament for the treatment of hepatitis B infection or related disorders. . The present invention provides a method of treating a hepatitis B infection or related disorder comprising administering to a patient infected with hepatitis B virus an effective amount of an oligomer or conjugate of the present invention that targets HBV. The present invention provides an in vivo or in vitro method for inhibiting HBV replication in HBV-infected cells, comprising administering to the cell an oligomer or conjugate of the present invention that targets HBV to inhibit HBV replication. provide. Examples of LNA oligomers targeting HBV (as disclosed in WO2011 / 47312) that can be used as oligomers of the invention (region A) are:

It is. Additional compounds are disclosed in Table 1 of WO2011 / 47312, as well as in WO2011 / 052911, WO2012 / 145674, WO2012 / 145697, WO2013 / 003520, and WO2013 / 159109, incorporated herein by reference.

  RG-101 is a compound that targets miR-122, contains a GalNac conjugate, and is under development for the treatment of HCV by Regulus Therapeutics.

  ANGPTL3 (eg, NCBI BC007059.1 GI: 14712025 or BC058287.1 GI: 34849466) Angiopoietin-like 3-lipid, glucose, and protein that regulates energy metabolism. Humans with elevated levels of ANGPTL3 have an increased risk of premature heart attack, hyperlipidemia associated with increased arterial wall thickness, and multiple metabolic abnormalities such as insulin resistance. In contrast, humans with lower ANGPTL3 levels have lower LDLC and triglyceride levels and a lower risk of cardiovascular disease. The compounds of the invention that target ANGPTL3 can be used, for example, in the treatment of hyperlipidemia and related disorders, metabolic disorders, atherosclerosis, coronary heart disease, or insulin resistance. WO11085271, incorporated herein by reference, discloses oligonucleotide compounds targeting ANGPTL3 that can be incorporated into the conjugates of the invention.

  Glucagon receptor or GCGR (BC112041.1 GI: 85567507; L20316.1 GI: 405189): Glucagon is a hormone that stimulates the liver to counteract the action of insulin and produce glucose, particularly in type 2 diabetes . In patients with advanced diabetes, unregulated glucagon action leads to a significant increase in blood glucose levels. Thus, diminished glucagon action may have a significant glucose lowering effect in patients with severe diabetes. Furthermore, a decrease in GCGR produces more active glucagon-like peptide or GLP-1, which is a hormone that preserves pancreatic function and enhances insulin secretion. Compounds of the invention that target GCGR are, for example, in the treatment of insulin resistance, hyperglycemia, diabetes, eg, type 1 or type 2 diabetes, in preservation of pancreatic function, and to regulate blood glucose levels, Can be used. WO2007 / 134014 discloses oligonucleotide compounds targeting GCGR that can be incorporated into the conjugates of the invention.

  Fibroblast growth factor receptor 4 or FGFR4 (NCBI gene 2264-NC_000005.9 (176513906..176525143)) FGFR4 is expressed in the liver and adipose tissue and reduces the body's ability to store fat while at the same time It has been shown to increase combustion and energy consumption. Many anti-obesity drugs act on the brain to suppress appetite and generally lead to CNS side effects. Compounds of the present invention that target FGFR4 are, for example, treatment of insulin resistance, hyperglycemia, diabetes, eg type 1 or type 2 diabetes, preservation of obesity (eg when used in combination with an appetite suppressant) , Weight loss, and insulin sensitivity, diabetes, eg, improvement of type 1 or type 2 diabetes, and regulation of blood glucose levels. WO09046141 and WO12174476, incorporated herein by reference, disclose oligonucleotide compounds targeting FGFR4 that can be incorporated into the conjugates of the invention.

  Diacylglycerol acyltransferase-2 or DGAT-2 (NCBI gene ID 84649): a critical component in the synthesis of triglycerides. Inhibition of DGAT can reduce liver fat in patients with nonalcoholic steatohepatitis (NASH) and can also be used to treat type 2 diabetes and insulin resistance. The compounds of the invention targeting DGAT-2 can be used to treat NASH, to reduce liver fat, to treat diabetes, eg, type 2 diabetes, to treat insulin resistance. WO05019418 and WO2007136989, incorporated herein by reference, disclose oligonucleotide compounds targeting DGAT-2 that can be incorporated into the conjugates of the invention.

  Glucocorticoid receptor or GCCR (BC150257.1 GI: 152013043): Glucocorticoid hormones affect a variety of processes in the body, and excessive levels of glucocorticoid hormones are harmful to many of the tissues and organs in the body Can have an effect. Cushing's syndrome is a strange disease caused by long-term exposure to high levels of glucocorticoids. If untreated, patients with Cushing's syndrome may develop hypertension, diabetes, and immune dysfunction and have an increased risk of premature death. Although there are approved treatments for Cushing's syndrome, current medications are associated with serious side effects such as hypertension and diabetes, and the high unmet medical need for new therapies for these patients Sex remains. The compounds of the invention that target GCCR-2 can be used to treat Cushing's syndrome and related conditions (eg, those listed above). WO07035759 and WO2007136988, which are hereby incorporated by reference, disclose oligonucleotide compounds targeting GCCR that can be incorporated into the conjugates of the invention.

  Complement component C5 (M57729.1 GI: 179982): Although the complement system plays a central role in immunity as a protective mechanism for host defense, its dysregulation is notably associated with paroxysmal nocturnal hemoglobinuria (PNH), leading to serious life-threatening complications in a wide range of human diseases, including atypical hemolytic uremic syndrome (aHUS), myasthenia gravis, and optic neuromyelitis. Compounds of the invention that target complement component C5 can be used to treat one or more of these disorders. C5 is a genetically and clinically validated target; loss-of-function human mutations are associated with reduced immune defenses against certain infections, resulting from intravenous administration of anti-C5 monoclonal antibodies Treatment has shown clinical activity and tolerability in a number of diseases mediated by complement. Transmembrane serine protease 6 (Tmprss6) for the treatment of beta thalassemia and iron overload disorders.

  α-1 antitrypsin (AAT): (M11465.1 GI: 177826) is associated with liver disease. WO13142514, incorporated herein by reference, discloses oligonucleotide compounds targeting AAT that can be incorporated into the oligomers or conjugates of the invention. Compounds of the invention that target AAT reduce AIAT mRNA and protein expression and treat fibrosis, eg, AIATD-related liver disease, and lung disease, eg, AIATD-related lung disease, in individuals in need thereof It can be used in a method to make, ameliorate, prevent, slow down or stop progression.

  Transthyretin-TTR (BC005310.1 GI: 13529049): The oligomers of the present invention that target TTR are transthyretin amyloidosis or TTR amyloidosis (mutations that produce misfolded forms of TTR that gradually accumulate in tissues) The gene can be used to treat severe rare inherited diseases that are inherited in patients. In patients with TTR amyloidosis, both mutant and normal forms of TTR can develop as fibrils in tissues including the heart, peripheral nerves, and gastrointestinal tract. The presence of TTR fibrils interferes with the normal function of these tissues, and as TTR protein fibrils expand, more tissue damage occurs and the disease worsens. TTR is a carrier protein that transports thyroid hormone and retinol in the blood. In patients with TTR amyloidosis, both mutant and normal forms of TTR can develop as fibrils in tissues. The compounds of the present invention can be used to treat TTR amyloidosis. See Benson et al., Amyloid. 2010 Jun; 17 (2): 43-9 and Ackermann et al., Amyloid.2012 Jun; 19 Suppl 1: 43-4. Antisense compounds targeting TTR that can be used in the oligomers or conjugates of the present invention are disclosed in US8101743, WO11139917, and WO10017509, which are incorporated herein by reference.

  Aminolevulinate synthase-1 (ALAS-1) (BC011798.2 GI: 33877783; AK312566.1 GI: 164690365; NM_199166.2 GI: 362999012; NM_000688.5 GI: 362999011). ALAS1 is a validated target for the treatment of porphyria, for example for the treatment of hepatic porphyria, including acute intermittent porphyria (AIP). The compounds of the invention that target ALAS-1 can be used in the treatment of these disorders.

  Vascular endothelial growth factor or VEGF (gene ID 7422, human sequence: chromosome: 6; NC_000006.11 (43737946..43754224)). VEGF has been shown in cancer. The compounds of the invention that target VEGF can be used in the treatment of hyperproliferative disorders, such as cancer, eg, liver cancer.

  Table 1 provides a group of liver targets that can be targeted by the compounds of the present invention, and medical indications / disorders where such compounds can be used to treat (eg, those suffering from related disorders) (See Sehgal et al., Liver as a target for oligonucleotide therapeutics, J. of Hepatology 2013, In Press).

Sequences In some embodiments, the oligomer or first region thereof comprises a contiguous nucleotide sequence corresponding to the reverse complement of the nucleotide sequence present in the target nucleic acid (ie, the sequence targeted by the oligomer). Table 3 provides a group of mRNA and miRNA targets that are in the course of preclinical or clinical development using oligonucleotide compounds for related indications and are therefore suitable for targeting by the compounds of the present invention. To do.

  In some embodiments, the target is miR-122, Apo B-100, Apo CIII, PCSK9, CRP, KSP, VEGF, PLK1, miR-34, FGFR4, Factor IXa, Factor XI, TTR, GCCR, PTP -1B, GCGR, AAT, ALDH2, HAMP pathway, miR-33, apo (a), miR-7, miR-378, miR-21, Myc, miR-122, HCV genome, such as HCV 5'UTR or HCV NS5B RNA or NS3 RNA, TMPRSS6, antithrombin III, apo CIII, ANGPLT3, MTP, DGAT2, ALAS1, antithrombin III, serum amyloid A, and factor VII.

  In some embodiments, the contiguous nucleotide sequence contains no more than one mismatch when hybridized to the target sequence.

  In determining the degree of “complementarity” between an oligomer (or region thereof) of the present invention and a target region of a nucleic acid, eg, those disclosed herein, “complementarity” (“homology” or The degree of “identity” is the percentage of identity (or percentage of homology) between the sequence of the oligomer (or region thereof) and the sequence of the target region (or the reverse complement of the target region) that is optimally aligned with it. ). The percentage is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of consecutive monomers in the oligomer and multiplying by 100. In such a comparison, if a gap exists, it is preferred that such a gap is merely a mismatch, rather than an area where the number of monomers in the gap differs between the oligomer of the invention and the target region.

  As used herein, the terms “homologous” and “homology” are interchangeable with the terms “identity” and “identical”.

  The terms “corresponding to” and “corresponds to” refer to oligomeric nucleotide sequences (ie, nucleobases or base sequences) or contiguous nucleotide sequences (first region); (I) Refers to a comparison between an equivalent continuous nucleotide sequence of a further sequence selected from any of the partial sequences of the reverse complementary strand of the nucleic acid target. Nucleotide analogs are directly compared to their equivalent or corresponding nucleotides. The first sequence corresponding to the additional sequence of (i) or (ii) is typically identical to that sequence over the entire length of the first sequence (eg, a contiguous nucleotide sequence) or as described herein. As described, in some embodiments, the corresponding sequence is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, It may be at least 96% homologous, for example 100% homologous (identical).

  The terms “corresponding nucleotide analog” and “corresponding nucleotide” are intended to indicate that the nucleotides in the nucleotide analog and the naturally occurring nucleotide are identical. For example, when a 2-deoxyribose unit of nucleotides is linked to adenine, a “corresponding nucleotide analog” contains a pentose unit (different from 2-deoxyribose) linked to adenine.

  As used herein, the terms “reverse complementary strand”, “reverse complementary”, and “reverse complementarity” are interchangeable with the terms “complementary strand”, “complementary”, and “complementarity”. It is.

  Thus, the continuous nucleobase sequence of the oligomer (first region or first region and second region) can be complementary to the target, eg, those mentioned herein.

  In some embodiments, the first region or the first region and the second region are mRNA targets, eg, those mentioned herein, eg, Apo B-100 (NM — 000384.2 GI: 105990531) or PCSK9 ( NM_174936.3 GI: 299523249) to form a single continuous nucleobase sequence complementary to the region.

Length Oligomers have a total length of 8 consecutive nucleotides, 9 consecutive nucleotides, 10 consecutive nucleotides, 11 consecutive nucleotides, 12 consecutive nucleotides, 13 consecutive nucleotides, 14 consecutive nucleotides, 15 consecutive nucleotides, 16 consecutive nucleotides Long, 17 contiguous nucleotide length, 18 contiguous nucleotide length, 19 contiguous nucleotide length, 20 contiguous nucleotide length, 21 contiguous nucleotide length, 22 contiguous nucleotide length, 23 contiguous nucleotide length, 24 contiguous nucleotide length, 25 contiguous nucleotide length, 26 contiguous nucleotide Long, 27 contiguous nucleotide length, 28 contiguous nucleotide length, 29 contiguous nucleotide length, 30 contiguous nucleotide length, 31 contiguous nucleotide length, 32 contiguous nucleotide length, 33 contiguous nucleotide length, 34 contiguous nucleotide length, and 35 contiguous nucleotide length Contains or is an array It may be.

  In some embodiments, the oligomer has a total length of 10-22 contiguous nucleotides, such as 12-18 contiguous nucleotides, such as 13-17 contiguous nucleotides, or 12-16 contiguous nucleotides, such as 13 contiguous nucleotides, It comprises or consists of a contiguous nucleotide sequence of 14 contiguous nucleotide lengths, 15 contiguous nucleotide lengths, 16 contiguous nucleotide lengths.

  In some embodiments, the oligomer comprises or consists of a contiguous nucleotide sequence of a total of 10 contiguous nucleotides, 11 contiguous nucleotides, 12 contiguous nucleotides, 13 contiguous nucleotides, or 14 contiguous nucleotides.

  In some embodiments, the oligomer according to the invention consists of no more than 22, such as no more than 20, such as no more than 18, such as 15, 16, or 17 nucleotides. In some embodiments, the oligomer according to the invention comprises less than 20 nucleotides. When a range is given for the length of the oligomer or contiguous nucleotide sequence, it includes the lower and upper lengths provided within the range, e.g. 10-30 includes both 10 and 30 It should be understood that

Nucleoside and Nucleoside Analogs As used herein, the term “nucleotide” refers to a sugar moiety (or analog thereof), a base moiety, and a covalently linked group (linking group), eg, phosphate type. Or a glycoside comprising a phosphorothioate-type internucleotide linking group, including naturally occurring nucleotides, such as DNA or RNA, and modified sugars and / or herein referred to as “nucleotide analogs” and / or Or both non-naturally occurring nucleotides containing a base moiety. Herein, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit.

  In the context of the present invention, it will be appreciated that the terms nucleoside and nucleotide are used to refer to both naturally occurring nucleotides / nucleosides, such as DNA and RNA, and nucleotide / nucleoside analogs.

  In the biochemical domain, the term “nucleoside” is commonly used to refer to a glycoside comprising a sugar moiety and a base moiety, and thus is covalently linked by an internucleoside linkage between oligomeric nucleotides. Can be used when referring to a given nucleotide unit. In the area of biotechnology, the term “nucleotide” is often used to refer to a nucleic acid monomer or unit, and thus, for oligonucleotides, it can refer to a base, eg, a “nucleotide sequence”, typically Refers to the nucleobase sequence (ie, the presence of sugar backbone and internucleoside linkages is potential). Similarly, particularly in the case of oligonucleotides in which one or more of the internucleoside linking groups are modified, the term “nucleotide” can refer to a “nucleoside”, eg, the presence of a linkage between nucleosides or Even when specifying a property, the term “nucleotide” may be used.

  As one skilled in the art will recognize, the 5 ′ terminal nucleotide of an oligonucleotide may or may not include a 5 ′ terminal group, but does not include a 5 ′ internucleoside linking group.

  Non-naturally occurring nucleotides include nucleotides having modified sugar moieties, such as bicyclic nucleotides, or 2 ′ modified nucleotides, such as 2 ′ substituted nucleotides.

スキーム1 “Nucleotide analogues” are variants of natural nucleotides, eg, DNA nucleotides or RNA nucleotides, by modification of sugar and / or base moieties. Analogs, in principle, are only “silent” or, with respect to oligonucleotides, are “equivalent” to natural nucleotides, ie, for the manner in which the oligonucleotide functions to inhibit target gene expression. It may not have a functional effect. However, such “equivalent” analogs are, for example, easier or cheaper to manufacture, or more stable to storage or manufacturing conditions, or represent a tag or label. In some cases, it may be useful. Preferably, however, the analog is expressed, for example, by increasing binding affinity for the target and / or increasing resistance to intracellular nucleases and / or increasing ease of transport into the cell. It will have a functional effect on the way the oligomer functions to inhibit Specific examples of nucleoside analogues are described, for example, by Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293-213. And is described in Scheme 1.

Scheme 1

  Thus, an oligomer may comprise or consist of a simple sequence of naturally occurring nucleotides, preferably 2'-deoxynucleotides (commonly referred to herein as "DNA"), but ribonucleotides (present It may also comprise or consist of a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, ie, nucleotide analogs, commonly referred to herein as “RNA”). is there. Such nucleotide analogs can optionally enhance the affinity of the oligomer for the target sequence.

  Examples of suitable preferred nucleotide analogues are provided by or referenced in WO2007 / 031091. Other nucleotide analogs that can be used in the oligomers of the invention include tricyclic nucleic acids, see, eg, WO2013154798 and WO2013154798, which are incorporated herein by reference.

  Incorporation of affinity-enhancing nucleotide analogs, such as LNA or 2′-substituted sugars into oligomers, can allow for a reduction in the size of the specifically binding oligomers and non-specific or abnormal binding It is also possible to reduce the upper limit on the size of the oligomer before it occurs.

  Oligomeric compounds, such as antisense oligonucleotides, such as the compounds mentioned herein, such as region A, and in some optional embodiments, region B contains one or more nucleosides that are modified at the sugar group You may do it. Such sugar-modified nucleosides (nucleoside analogs) can confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compound. In some embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety.

  In some embodiments, the oligomer or first region thereof is at least 1, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, At least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, Include at least 22, at least 23, at least 24, or 25 nucleoside analogs, eg, sugar-modified nucleoside analogs.

Bicyclic nucleoside analogs include nucleoside analogs that contain a bridge (or biradical) that connects the second and fourth carbons of the ribose ring (C4 ^* -C2 ^* bridge or biradical). The presence of a biradical between the 2nd and 4th carbon locks the ribose into the 3 ′ endo- (N-type) conformation, and thus a bicyclic nucleoside analog with a C2 ^* -C4 ^* biradical is Often referred to as locked nucleic acid (LNA). In some embodiments, the nucleoside analog is (optionally) selected from the group consisting of a bicyclic nucleoside analog (eg, LNA) and / or a 2′-substituted nucleoside analog, such as (optionally Independently) 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-DNA units, 2'-AP, 2'-FANA, 2 '-(3-hydroxy) propyl, and 2'-fluoro-DNA units, and / or other (optional) sugar-modified nucleoside analogs such as morpholino, peptide nucleic acid (PNA), CeNA, unlinked nucleic acid (UNA), hexitol nucleic acid (HNA), bicyclo -Selected from the group consisting of HNA (see eg WO2009 / 100320). In some embodiments, the nucleoside analog increases the affinity of the first region for its target nucleic acid (or complementary DNA or RNA sequence).

  In some embodiments, the oligomer comprises at least one bicyclic nucleotide analog, eg, LNA. In some embodiments, the first region comprises at least one bicyclic nucleoside analog (eg, LNA) and / or a 2 ′ substituted nucleoside analog. In some embodiments, the nucleoside analogs present in the oligomer all contain the same sugar modification. In some embodiments, the at least one nucleoside analog present in the first region is a bicyclic nucleoside analog, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, for example, all nucleoside analogs (Other than the DNA nucleoside and / or RNA nucleoside of region B) is a sugar-modified nucleoside analog such as a bicyclic nucleoside analog such as LNA such as β-DX-LNA or α-LX-LNA (where X is Or other LNAs disclosed herein, for example, but not limited to (R / S) cET, cMOE, or Is 5'-Me-LNA.

Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (eg, 5 ′ and 2 ′ substituents); non-rings to form bicyclic nucleic acids (BNA) Bridge of geminal ring atoms; substitution of ribosyl ring oxygen atoms with S, N (R), or C (R ₁ ) (R ₂ ) (R = H, C ₁ -C ₂ alkyl, or protecting groups); and Is included. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (published on 8/21/08 for other disclosed 5 ', 2'-bis substituted nucleosides Published PCT international application WO2008 / 101157), substitution of the ribosyl ring oxygen atom to S and further substitution at the 2 ′ position (published US patent application US2005 / 0130923 published 16 June 2005) Or 5 'substitution of BNA (see, for example, PCT International Application WO2007 / 134181 published on 11/22/07, where LNA is substituted by, for example, a 5'-methyl group or a 5'-vinyl group) Is included.

Examples of nucleosides having modified sugar moieties include, but are not limited to, 5′-vinyl substituents, 5′-methyl substituents (R or S), 4′-S substituents, 2′-F substituents, 2'-OCH ₃ substituents, and 2'-O (CH ₂₎ includes nucleosides that include a 2OCH ₃ substituents. Substituents at the 2 ′ position are allyl, amino, azide, thio, O-allyl, OC ₁ -C ₁₀ alkyl, OCF ₃ , O (CH ₂ ) ₂ SCH ₃ , O (CH ₂ ) ₂ -ON (Rm) (Rn), and O—CH ₂ —C (═O) —N (Rm) (Rn) [Rm and Rn are each independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl. ] Can also be selected.

As used herein, “bicyclic nucleoside” refers to a modified nucleoside that includes a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides that include a bridge between the 4 ′ and 2 ′ ribosyl ring atoms. In some embodiments, the compounds provided herein include one or more bicyclic nucleosides, where the bridge comprises a 4′-2 ′ bicyclic nucleoside. Examples of such 4′-2 ′ bicyclic nucleosides include, but are not limited to, any of the following formulas: 4 ′-(CH ₂ ) —O-2 ′ (LNA) _{; 4 '- (CH 2)} -S-2'; 4 '- (CH 2) 2 -O-2'(ENA);4'-CH (CH 3) -O-2 ', and 4'-CH (CH ₂ OCH ₃ ) -O-2 ^* , and analogs thereof (see US Pat. No. 7,399,845 issued July 15, 2008); 4′-C (CH ₃ ) (CH ₃ ) — O-2 ′ and analogs thereof (see published PCT international application WO2009 / 006478 published on January 8, 2009); 4′-CH ₂ —N (OCH ₃ ) -2 ′ and analogs thereof (See published PCT international application WO2008 / 150729 published on 11 December 2008); 4'-CH ₂ -ON (CH ₃ ) -2 '(published 2 September 2004) Published US patent application US2004 / 0171570); 4′-CH ₂ —N (R) —O-2 ′, where R is H, C ₁ -C ₁₀ alkyl, or a protecting group (2008) year see issued US Patent No. 7,427,672 on September _{23); 4'-CH 2 -C (} H) (CH 3) -2 '(Chat topadhyaya, et al, J. Org. Chem., 2009, 74, 118-134); and 4'-CH ₂ -C (= CH ₂ ) -2 'and analogs (published on 8 December 2008) Published PCT international application WO 2008/154401). For example, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97,5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10033-10039; Srivastava et al., J. Am. Chem. Soc, 129 (26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem Biol, 2001, 8, 1-7; Oram et al, Curr. Opinion Mol. Ther., 2001, 3, 239-243; US Pat. Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499 7,034,133, 6,525,191, 7,399,845; published PCT international applications WO2004 / 106356, WO94 / 14226, WO2005 / 021570, and WO2007 / 134181; US Patent Publication Nos. US2004 / 0171570, US2007 / 0287831, and US2008 And US Patent Application Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT International Application No. PCT / See also US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922. Each of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, such as aL-ribofuranose and β-D-ribofuranose (WO99 / 25 March 25, 1999). PCT International Application PCT DK98 / 00393 published as 14226).

In some embodiments, the bicyclic sugar moiety of the BNA nucleoside includes, but is not limited to, a compound having at least one bridge between the 4 ′ and 2 ′ positions of the pentofuranosyl sugar moiety. Not. Such bridges are independently-[CiR _a XR _b )] ,,-, -C (R _a ) = C (R _b )-, -C (R _a ) = N-, -C (= NR _a )-, -C (= O)-, -C (= S)-, -O-, -Si (R _a ) _2- , -S (= O) _x- , and -N (Ra)-[ Where x is 0, 1, or 2; n is 1, 2, 3, or 4; R _a and R _b are each independently H, protecting group, hydroxyl, C ₁ − C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -Ci ₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -Ci ₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ Aryl, substituted C ₅ -C ₂₀ aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C ₅ -C ₇ alicyclic radical, substituted C ₅ -C ₇ alicyclic radical , Halogen, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ , acyl (C (= O) -H), substituted acyl, CN, sulfonyl (S (= O) ₂ -J ₁ ), or be a sulfoxyl _{(S (= O) -J 1} ); J 1 and J ₂ are each Independently, H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, acyl (C (= O) -H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C ₁₂ aminoalkyl A substituted C ₁ -C ₁₂ aminoalkyl, or a protecting group] containing 1 or 2 to 4 linked groups selected independently.

In some embodiments, the bridge of the bicyclic sugar moiety is-[C (R _a ) (Rb)] _n -,-[C (R _a ) (R _b )] _n -O-, -C (R _a R _b ) —N (R) —O—, or —C (R _a R _b ) —ON (R) —. In some embodiments, the bridge is 4'-CH ₂ -2 ', 4'-(CH ₂ ) ₂ -2 ', 4'-(CH ₂ ) ₃ -2 ', 4'-CH ₂ -O- 2 ', 4 ^* -(CH ₂ ) 2-O-2', 4'-CH ₂ -ON (R) -2 ', and 4'-CH ₂ -N (R) -O-2'-[formula In which each R is independently H, a protecting group, or C ₁ -C ₁₂ alkyl].

In some embodiments, the bicyclic nucleoside is further defined by isomeric configuration. For example, a nucleoside containing a 4′-2 ′ methylene-oxy bridge can be in the aL or β-D configuration. Previously, aL-methyleneoxy (4'-CH ₂ -O-2 ') BNA was incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365). -6372).

式中、Bxは、塩基部分であり、Rは、独立に、H、保護基、またはC₁-C₂アルキルである。 In some embodiments, the bicyclic nucleoside includes (A) aL-methyleneoxy (4′-CH ₂ —O-2 ′) BNA, (B) β-D-methylene, as illustrated below. Oxy (4'-CH ₂ -O-2 ') BNA, (C) Ethyleneoxy (4'-(CH ₂ ) ₂ -O-2 ') BNA, (D) Aminooxy (4'-CH ₂ -ON) (R) -2 ') BNA, (E) Oxyamino (4'-CH ₂ -N (R) -O-2') BNA, (F) Methyl (methyleneoxy) (4'-CH (CH ₃ ) -O-2 ') BNA, (G) Methylene-thio (4'-CH ₂ -S-2') BNA, (H) Methylene-amino (4'-CH ₂ -N (R) -2 ') BNA , (I) methyl carbocyclic (4′-CH ₂ —CH (CH ₃ ) -2 ′) BNA, and (J) propylene carbocyclic (4 ′-(CH ₂ ) ₃ -2 ′) BNA However, it is not limited to these.

Where Bx is a base moiety and R is independently H, a protecting group, or C ₁ -C ₂ alkyl.

式中、Bxは、複素環式塩基部分であり；
-Q_a-Q_b-Q_c-は、-CH₂-N(Rc)-CH₂-、-C(=O)-N(R_c)-CH₂-、-CH₂-O-N(Rc)-、-CH₂-N(Rc)-O-、または-N(Rc)-O-CH₂であり；
R_cは、C₁-C₁₂アルキルまたはアミノ保護基であり；かつ
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体への共有結合性の付着である。 In certain embodiments, the bicyclic nucleoside has the formula I:

Where Bx is a heterocyclic base moiety;
-Q _a -Q _b -Q _c -is -CH ₂ -N (Rc) -CH _2- , -C (= O) -N (R _c ) -CH _2- , -CH ₂ -ON (Rc) -, -CH ₂ -N (Rc) -O-, or -N (Rc) -O-CH ₂ ;
R _c is a C ₁ -C ₁₂ alkyl or amino protecting group; and
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium.

式中、Bxは、複素環式塩基部分であり；
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体への共有結合性の付着であり；Z_aは、C₁-C₆アルキル、C₂-C₆アルケニル、C₂-C₆アルキニル、置換型C₁-C₆アルキル、置換型C₂-C₆アルケニル、置換型C₂-C₆アルキニル、アシル、置換型アシル、置換型アミド、チオール、または置換型チオである。 In some embodiments, the bicyclic nucleoside has the formula II:

Where Bx is a heterocyclic base moiety;
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; Z _a is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, acyl, substituted acyl, Substituted amide, thiol, or substituted thio.

In some embodiments, each substituted group is independently halogen, oxo, hydroxyl, OJ _c , NJ _d , SJ _c , N ₃ , OC (= X) J _c , and NJ _e C (= X). It is mono- or polysubstituted by a substituent independently selected from NJ _c J _d . J _c , J _d , and J _e are each independently H, C ₁ -C ₆ alkyl, or substituted C ₁ -C ₆ alkyl, and X is O or NJ _C.

式中、Bxは、複素環式塩基部分であり；
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体への共有結合性の付着であり；
R_dは、C₁-C₆アルキル、C₂-C₆アルケニル、C₂-C₆アルキニル、置換型C₁-C₆アルキル、置換型C₂-C₆アルケニル、置換型C₂-C₆アルキニル、または置換型アシル（C(=O)-）である。 In some embodiments, the bicyclic nucleoside has the formula III:

Where Bx is a heterocyclic base moiety;
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
R _d is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ Alkynyl or substituted acyl (C (= O)-).

式中、Bxは、複素環式塩基部分であり；
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体への共有結合性の付着であり；
R_dは、C₁-C₆アルキル、置換型C₁-C₆アルキル、C₂-C₆アルケニル、置換型C₂-C₆アルケニル、C₂-C₆アルキニル、置換型C₂-C₆アルキニルであり；q_b、q_c、およびq_dは、各々独立に、H、ハロゲン、C₁-C₆アルキル、置換型C₁-C₆アルキル、C₂-Ceアルケニル、置換型C₂-C₆アルケニル、C₂-C₆アルキニル、または置換型C₂-C6アルキニル、C₁-C₆アルコキシル、置換型Q-C₆アルコキシル、アシル、置換型アシル、C₁-C₆アミノアルキル、または置換型C₁-C₆アミノアルキルである。 In some embodiments, the bicyclic nucleoside has the formula IV:

Where Bx is a heterocyclic base moiety;
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;
R _d is C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ Alkynyl; q _b , q _c , and q _d are each independently H, halogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -Ce alkenyl, substituted C _2- C ₆ alkenyl, C ₂ -C ₆ alkynyl, or substituted C ₂ -C 6 alkynyl, C ₁ -C ₆ alkoxyl, substituted QC ₆ alkoxyl, acyl, substituted acyl, C ₁ -C ₆ aminoalkyl, or substituted C ₁ -C ₆ aminoalkyl.

式中、Bxは、複素環式塩基部分であり；
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体への共有結合性の付着であり；q_a、q_b、q_c、およびq_fは、各々独立に、水素、ハロゲン、C₁-C₁₂アルキル、置換型C₁-C₁₂アルキル、C₂-C₁₂アルケニル、置換型C₂-C₁₂アルケニル、C₂-C₁₂アルキニル、置換型C₂-C₁₂アルキニル、C₁-C₁₂アルコキシ、置換型C₁-C₁₂アルコキシ、OJ_j、SJ_j、SOJ_j、SO₂J_j、NJ_jJ_k、N₃、CN、C(=O)OJ_j、C(=O)NJ_jJ_k、C(=O)J_j、O-C(=O)NJ_jJ_k、N(H)C(=NH)NJ_jJ_k、N(H)C(=O)NJ_jJ_k、もしくはN(H)C(=S)NJ_jJ_kであるか；またはq_eおよびq_fは、共に、=C(q_g)(q_h)であり；q_gおよびq_hは、各々独立に、H、ハロゲン、C₁-C₁₂アルキル、または置換型C₁-C₁₂アルキルである。 In some embodiments, the bicyclic nucleoside has the formula V:

Where Bx is a heterocyclic base moiety;
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; q _a , q _b , q _c , And q _f are each independently hydrogen, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxy, substituted C ₁ -C _{12 alkoxy, OJ j, SJ j, SOJ} j, SO 2 J j, NJ j J k, N 3, CN, C (= O) OJ _j , C (= O) NJ _j J _k , C (= O) J _j , OC (= O) NJ _j J _k , N (H) C (= NH) NJ _j J _k , N (H) C (= O) NJ _j J _k , or N (H) C (= S) NJ _j J _k ; or q _e and q _f are both = C (q _g ) be (q _h); q _g and q _h are each independently, H, halogen, a C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

Methyleneoxy _{(4'-CH 2 -O-2} ') BNA monomers adenine, cytosine, guanine, 5-methyl - cytosine, thymine, and synthesis and preparation of uracil are described along with their oligomerization, and nucleic acid recognition properties (See, for example, Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNA and its preparation are also described in WO98 / 39352 and WO99 / 4226.

Analogs of methyleneoxy (4'-CH ₂ -O-2 ') BNA, methyleneoxy (4'-CH ₂ -O-2') BNA, and 2'-thio-BNA have also been prepared (eg, Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes containing locked nucleoside analogs as substrates for nucleic acid polymerases has also been described (see, for example, Wengel et al., WO99 / 14226). In addition, the synthesis of 2′-amino-BNA, a novel conformationally restricted high affinity oligonucleotide analog, has been described in the art (see, eg, Singh et al., J. Org. Chem., 1998, 63, 10033-10039). In addition, 2'-amino-BNA and 2'-methylamino-BNA have been prepared and their duplex thermal stability with complementary RNA and DNA strands has been previously reported.

式中、Bxは、複素環式塩基部分であり；
T_aおよびT_bは、各々独立に、H、ヒドロキシル保護基、コンジュゲート基、反応性リン基、リン部分、または支持媒体へ共有結合性の付着であり；qj、qj、q_k、およびqlは、各々独立に、H、ハロゲン、C₁-C₁₂アルキル、置換型C₁-C₁₂アルキル、C₂-C₁₂アルケニル、置換型C₂-C₁₂アルケニル、C₂-C₁₂アルキニル、置換型C₂-C₁₂アルキニル、C₁-C₁₂アルコキシル、置換型C₂-C₁₂アルコキシル、OJ_j、SJ_j、SOJ_j、SO₂J_j、NJ_jJ_k、N₃、CN、C(=O)OJ_j、C(=O)NJ_jJ_k、C(=O)J_j、O-C(=O)NJ_jJ_k、N(H)C(=NH)NJ_jJ_k、N(H)C(=O)NJ_jJ_k、または(H)C(=S)NJ_jJ_kであり；かつqiおよびq_jまたはqlおよびq_kは、共に、=C(q_g)(q_h)［式中、q_gおよびq_hは、各々独立に、H、ハロゲン、C₁-C₁₂アルキル、または置換型C₁-C₆アルキルである］である。 In some embodiments, the bicyclic nucleoside has the formula VI:

Where Bx is a heterocyclic base moiety;
T _a and T _b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium; qj, qj, q _k , and ql are each independently, H, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted type C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxyl, substituted C ₂ -C _{12 alkoxyl, OJ j, SJ j, SOJ} j, SO 2 J j, NJ j J k, N 3, CN, C ( = O) OJ _j , C (= O) NJ _j J _k , C (= O) J _j , OC (= O) NJ _j J _k , N (H) C (= NH) NJ _j J _k , N ( H) C (= O) NJ _j J _k , or (H) C (= S) NJ _j J _k ; and qi and q _j or ql and q _k are both = C (q _g ) (q _h ) wherein q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl, or substituted C ₁ -C ₆ alkyl.

Carbocyclic bicyclic nucleosides with 4 ′-(CH ₂ ) ₃ -2 ′ bridges and alkenyl analogs, bridges 4′-CH═CH—CH ₂ -2 ′ have been described (eg, Freier et al. al, Nucleic Acids Research, 1997, 25 (22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731-77'40). The synthesis and preparation of carbocyclic bicyclic nucleosides has also been described along with their oligomerization and biochemical studies (eg, Srivastava et al, J. Am. Chem. Soc. 2007, 129 (26), 8362. -8379).

  As used herein, “4 ′ to 2 ′ (4′-2 ′) bicyclic nucleoside” or “4 ′ to 2 ′ (4 ′ to 2 ′) bicyclic nucleoside” refers to 2 ′ A bicyclic nucleoside containing a furanose ring containing a bridge connecting a carbon atom and a 4 ′ carbon atom.

As used herein, “monocyclic nucleoside” refers to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In some embodiments, the sugar moiety or sugar moiety analog of the nucleoside may be modified or substituted at any position. As used herein, “2 ′ modified sugar” means a furanosyl sugar modified at the 2 ′ position. In some embodiments, such modifications include substituents selected from: halides, such as, but not limited to, substituted and unsubstituted alkoxy, substituted and non- Substituted thioalkyls, substituted and unsubstituted aminoalkyls, substituted and unsubstituted alkyls, substituted and unsubstituted allyls, and substituted and unsubstituted alkynyls. In some embodiments, the 2 ′ modification is O [(CH ₂ ) _n O] _m CH ₃ , O (CH ₂ ) ,, NH ₂ , O (CH ₂ ), CH ₃ , O (CH ₂ ), , ONH ₂ , OCH ₂ C (═O) N (H) CH ₃ , and O (CH ₂ ) nON [(CH ₂ ) _n CH ₃ ] 2 where n and m are 1 to about 10. ), But is not limited to these. Other 2 ′ substituents may also be selected from: C ₁ -C ₁₂ alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH ₃ ; _{Cl; Br; CN; CF 3} ; OCF 3; SOCH 3; SO 2 CH 3; ONO 2; NO 2; N 3; NH 2; heterocycloalkyl; heterocycloalkyl alkaryl; aminoalkyl amino; polyalkyl amino; substituted Type silyl; R; cleavage group; reporter group; intercalator; group for improving pharmacokinetic properties; and groups for improving the pharmacodynamic properties of antisense compounds, and others with similar properties Substituents. In some embodiments, the modified nucleoside comprises a 2′-MOE side chain (see, eg, Baker et al., J. Biol. Chem., 1997, 272, 1 1944-12000). Such 2′-MOE substitutions have improved binding affinity compared to unmodified nucleosides and other modified nucleosides such as 2′-O-methyl, O-propyl, and O-aminopropyl. It is described that it has. Oligonucleotides with 2-MOE substituents have also been shown to be antisense inhibitors of gene expression with promising features for use in vivo (eg, Martin, P., He / v. Chim Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997 , 16, 917-926).

式中、式Xの少なくとも1つの前記テトラヒドロピランヌクレオシド類似体のそれぞれについて独立に、
Bxは、複素環式塩基部分であり；
T₃およびT₄は、各々独立に、テトラヒドロピランヌクレオシド類似体をアンチセンス化合物と連結しているヌクレオシド間連結基であるか、またはT₃およびT₄の一方が、テトラヒドロピランヌクレオシド類似体をアンチセンス化合物と連結しているヌクレオシド間連結基であり、かつT₃およびT4の他方が、H、ヒドロキシル保護基、連結されたコンジュゲート基、または5'末端もしくは3'末端の基であり；q₁、q₂、q₃、q₄、q₅、q₆、およびq₇は、各々独立に、H、C₁-C₆アルキル、置換型C₁-C₆アルキル、C₂-C₆アルケニル、置換型C₂-C₆アルケニル、C₂-C₆アルキニル、または置換型C₂-C₆アルキニルであり；かつR₁およびR₂の一方は、水素であり、かつ他方は、ハロゲン、置換型または非置換型のアルコキシ、NJ、J₂、SJ、N₃、OC(=X)J₁、OC(=X)NJ₁J₂、NJ₃C(=X)NJ₁J₂、およびCN［式中、Xは、O、S、またはNJ₁であり、J₁、J₂、およびJ₃は、各々独立に、HまたはC₁-C₆アルキルである］より選択される。 As used herein, “modified tetrahydropyran nucleoside” or “modified THP nucleoside” refers to the substitution of a pentofuranosyl residue in a normal nucleoside with a 6-membered tetrahydropyran “sugar” (sugar substitute). Means a nucleoside. Modified THP nucleosides include hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (Leumann, CJ. Bioorg. And Med. Chem. (2002) 10: 841-854). Reference), what is referred to in the art as fluoro-HNA (F-HNA), or compounds having the formula X include, but are not limited to:
Formula X

Wherein for each of the at least one said tetrahydropyran nucleoside analogue of formula X independently
Bx is a heterocyclic base moiety;
T ₃ and T ₄ are each independently an internucleoside linking group that links a tetrahydropyran nucleoside analog to an antisense compound, or one of T ₃ and T ₄ is an anti-tetrahydropyran nucleoside analog. An internucleoside linking group linked to the sense compound, and the other of T ₃ and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a group at the 5 ′ end or 3 ′ end; q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , and q ₇ are each independently H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl , Substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, or substituted C ₂ -C ₆ alkynyl; and _one of R ₁ and R ₂ is hydrogen and the other is halogen, substituted Type or unsubstituted alkoxy, NJ, J ₂ , SJ, N ₃ , OC (= X) J ₁ , OC (= X) N J ₁ J ₂ , NJ ₃ C (= X) NJ ₁ J ₂ , and CN [wherein X is O, S, or NJ ₁ and J ₁ , J ₂ , and J ₃ are each independently , H or C ₁ -C ₆ alkyl].

In some embodiments, there is provided a modified THP nucleoside of formula X, wherein q _m , q _n , q _p , q _r , q _s , q _t , and q _u are each H. In some embodiments, at least one of q _m , q _n , q _p , q _r , q _s , q _t , and q _u is other than H. In some embodiments, at least one of the _{q m, q n, q p} , q r, q s, q t, and q _u are methyl. In some embodiments, provided is a THP nucleoside of Formula X wherein one of R ₁ and R ₂ is F. In some embodiments, R ₁ is fluoro, R ₂ is H, R ₁ is methoxy, R ₂ is H, R ₁ is methoxyethoxy, and R ₂ is H. is there.

As used herein, “2 ′ modified” or “2 ′ substituted” refers to a nucleoside containing a sugar containing a substituent other than H or OH at the 2 ′ position. 2′-modified nucleosides include non-bridged 2 ′ substituents such as allyl, amino, azide, thio, O-allyl and OC ₁ -C ₁₀ alkyl, —OCF ₃ , O— (CH ₂ ) ₂ —O— CH ₃ , 2'-O (CH ₂ ) ₂ SCH ₃ , O- (CH ₂ ) ₂ -ON (R _m ) (R _n ), or O-CH ₂ -C (= O) -N (R _m ) Including, but not limited to, a nucleoside having (R _n ), wherein R _m and R, are each independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl. . The 2 ′ modified nucleoside may further comprise other modifications, eg, at other positions on the sugar and / or at the nucleobase.

  As used herein, “2′-F” refers to a sugar containing a fluoro group at the 2 ′ position.

As used herein, “2′-OMe” or “2′-OCH ₃ ” or “2′-O-methyl” refers to a sugar containing an —OCH ₃ group at the 2 ′ position of the sugar ring, respectively. A nucleoside containing

  As used herein, “oligonucleotide” refers to a compound comprising a number of linked nucleosides.

  In some embodiments, one or more of the multiple nucleosides is modified. In some embodiments, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

  Many other bicyclosaccharide and tricyclosaccharide alternative ring systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art (eg, review paper: Leumann, JC, Bioorganic and Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity. Methods for the preparation of modified sugars are well known to those skilled in the art. In nucleotides having modified sugar moieties, the nucleobase moiety (natural, modified, or combinations thereof) is maintained for hybridization with the appropriate nucleic acid target.

  In some embodiments, the antisense compound comprises one or more nucleotides with modified sugar moieties. In some embodiments, the modified sugar moiety is 2'-MOE. In some embodiments, the 2′-MOE modified nucleotide is placed in the gapmer motif. In some embodiments, the modified sugar moiety is cEt. In some embodiments, the cEt modified nucleotide is placed throughout the wing of the gapmer motif.

In some embodiments, in BNA (LNA), R ^{4 *} and R ^{2 *} both represent a biradical —O—CH (CH ₂ OCH ₃ ) — in either the R or S configuration (2 ′ O-methoxyethyl bicyclic nucleic acid-Seth at al., 2010, J. Org. Chem).

In some embodiments, in BNA (LNA), R ^{4 *} and R ^{2 *} both represent a biradical —O—CH— (CH ₂ CH ₃ ) — in either the R or S configuration (2 'O-ethyl bicyclic nucleic acid-Seth at al., 2010, J. Org. Chem).

In some embodiments, in BNA (LNA), R ^{4 *} and R ^{2 *} both represent a biradical —O—CH— (CH ₃ ) — in either the R or S configuration. In some embodiments, R ^{4 *} and R ^{2 *} both represent the biradical —O—CH ₂ —O—CH ₂ — (Seth at al., 2010, J. Org. Chem).

In some embodiments, in BNA (LNA), R ^{4 *} and R ^{2 *} both represent the biradical —O—NR—CH ₃ — (Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA unit has a structure selected from the following group:

  Thus, an oligomer may comprise or consist of a simple sequence of naturally occurring nucleotides, preferably 2′-deoxynucleotides (commonly referred to herein as “DNA”), although ribonucleotides ( May be comprised of or consist of a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, ie, nucleotide analogs, commonly referred to herein as "RNA") It is. Such nucleotide analogs can optionally enhance the affinity of the oligomer for the target sequence.

  Incorporation of affinity-enhancing nucleotide analogs, such as BNA (eg, LNA) or 2′-substituted sugars, into oligomers can allow for a reduction in the size of the specifically bound oligomer and is non-specific Alternatively, the upper limit on the size of the oligomer can be reduced before abnormal binding occurs.

  In some embodiments, the oligomer comprises at least one nucleoside analog. In some embodiments, the oligomer comprises at least 2 nucleotide analogues. In some embodiments, the oligomer comprises 3-8 nucleotide analogs, eg, 6 or 7 nucleotide analogs. In presently most preferred embodiments, at least one of the nucleotide analogs is a BNA, such as a locked nucleic acid (LNA); for example, at least 3 or at least 4 or at least 5 of the nucleotide analogs or At least 6 or at least 7 or 8 can be BNA, eg, LNA. In some embodiments, all nucleotide analogs can be BNA, eg, LNA.

When referring to a preferred nucleotide sequence motif or nucleotide sequence consisting only of nucleotides, the oligomer of the invention defined by that sequence replaces the corresponding nucleotide analog instead of one or more of the nucleotides present in the sequence, for example, oligomers / target duplex duplex stability / T BNA units or other nucleotide analogues to raise the _m (i.e., affinity enhancing nucleotide analogues) that may contain a recognized I will.

  Preferred nucleotide analogs are LNAs such as oxy-LNA (eg β-D-oxy-LNA and α-L-oxy-LNA) and / or amino-LNA (eg β-D-amino-LNA and α -L-amino-LNA) and / or thio-LNA (eg β-D-thio-LNA and α-L-thio-LNA) and / or ENA (eg β-D-ENA and α-L-ENA) ). Most preferred is β-D-oxy-LNA.

  In some embodiments, nucleotide analogs present in the oligomers of the invention include, for example, 2′-O-alkyl-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, BNA units, For example, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA units (intercalating nucleic acids-Christensen, 2002. Nucl. Acids. Res. 2002, incorporated herein by reference. 30: 4918-4925), and 2'MOE units: selected more independently. In some embodiments, only one of the above types of nucleotide analogs is present in the oligomer of the invention, eg, in its first region or continuous nucleotide sequence.

  In some embodiments, the nucleotide analog is 2′-O-methoxyethyl-RNA (2′MOE), 2′-fluoro-DNA monomer, or LNA nucleotide analog, and thus the oligonucleotides of the invention are May include nucleotide analogs independently selected from these three types of analogs, or may include only one type of analog selected from the three types. In some embodiments, at least one of the nucleotide analogs is 2'-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 Or 10 are 2'-MOE-RNA nucleotide units. In some embodiments, at least one of the nucleotide analogs is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 Or 10 are 2'-fluoro-DNA nucleotide units.

  In some embodiments, the oligomer according to the invention comprises at least one BNA, such as a locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, Or 8 BNA / LNA units, eg 3-7 or 4-8 BNA / LNA units, or 3, 4, 5, 6 or 7 BNA / LNA units. In some embodiments, all nucleotide analogs are BNA, eg, LNA. In some embodiments, the oligomer may comprise both β-D-oxy-LNA and one or more of the following LNA units: either β-D configuration or α-L configuration or combinations thereof Thio-LNA, amino-LNA, oxy-LNA, and / or ENA. In some embodiments, every BNA, eg, LNA cytosine unit is 5'-methyl-cytosine. In some embodiments of the invention, the oligomer (eg, the first region, optionally the second region) may comprise both BNA and LNA units and DNA units. In some embodiments, the sum of LNA units and DNA units is 10-25, such as 10-24, preferably 10-20, such as 10-18, such as 12-16. In some embodiments of the invention, the nucleotide sequence of the oligomer or its first region, eg, a contiguous nucleotide sequence, consists of at least one BNA, eg, LNA, and the remaining nucleotide units are DNA units. In some embodiments, the oligomer or first region thereof is only BNA, eg, LNA, nucleotide analogs, and naturally occurring nucleotides (eg, RNA nucleotides or DNA nucleotides, most preferably DNA nucleotides), optionally In combination with modified internucleotide linkages, such as phosphorothioates.

  The term “nucleobase” refers to the base portion of a nucleotide and includes both naturally occurring and non-naturally occurring variants. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also their heterocyclic analogs and tautomers. It will be appreciated that the DNA or RNA nucleoside of region B may have naturally occurring and / or non-naturally occurring nucleobases.

  Examples of nucleobases include adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2 -Includes, but is not limited to, aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine. In some embodiments, the nucleobase can be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, uracil, 5-methylcytosine. In some embodiments, the nucleobase can be independently selected from the group consisting of adenine, guanine, cytosine, thymidine, and 5-methylcytosine.

  In some embodiments, at least one of the nucleobases present in the oligomer is 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2- A modified nucleobase selected from the group consisting of aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

LNA
The term “LNA” refers to a bicyclic nucleoside analog that contains a C2 ^* -C4 ^* biradical (bridge) and is known as a “locked nucleic acid”. It may refer to an LNA monomer or, when used in reference to an “LNA oligonucleotide”, an LNA refers to an oligonucleotide containing one or more such bicyclic nucleotide analogs. In some aspects, the bicyclic nucleoside analogs are LNA nucleotides, and thus these terms can be used interchangeably, and in such embodiments, both are C2 ′ and C4 of the ribose sugar ring. Characterized by the presence of a linker group (for example, a bridge) between.

式中、Yは、-O-、-CH₂O-、-S-、-NH-、N(R^e)、および／または-CH₂-からなる群より選択され；

は、ヌクレオチド間連結、R^H、末端基、または保護基の中から独立に選択され；Bは、天然または非天然のヌクレオチド塩基部分（核酸塩基）を構成し、R^Hは、水素およびC_1-4-アルキルより選択され；R^a、R^b、R^c、R^d、およびR^eは、任意で独立に、水素、置換されていてもよいC_1-12-アルキル、置換されていてもよいC_2-12-アルケニル、置換されていてもよいC_2-12-アルキニル、ヒドロキシ、C_1-12-アルコキシ、C_2-12-アルコキシアルキル、C_2-12-アルケニルオキシ、カルボキシ、C_1-12-アルコキシカルボニル、C_1-12-アルキルカルボニル、ホルミル、アリール、アリールオキシ-カルボニル、アリールオキシ、アリールカルボニル、ヘテロアリール、ヘテロ-アリールオキシ-カルボニル、ヘテロアリールオキシ、ヘテロアリールカルボニル、アミノ、モノ-およびジ(C_1-6-アルキル)アミノ、カルバモイル、モノ-およびジ(C_1-6-アルキル)-アミノ-カルボニル、アミノ-C_1-6-アルキル-アミノカルボニル、モノ-およびジ(C_1-6-アルキル)アミノ-C_1-6-アルキル-アミノカルボニル、C_1-6-アルキル-カルボニルアミノ、カルバミド、C_1-6-アルカノイルオキシ、スルホノ（sulphono）、C_1-6-アルキルスルホニルオキシ、ニトロ、アジド、スルファニル、C_1-6-アルキルチオ、ハロゲン、DNAインターカレーター、光化学的活性を有する基、熱化学的活性を有する基、キレート基、レポーター基、ならびにリガンドからなる群より選択され、ここで、アリールおよびヘテロアリールは、置換されていてもよく、2個のジェミナル置換基R^aおよびR^bは、共に、置換されていてもよいメチレン（=CH₂）を表し得；R^Hは、水素およびC_1-4-アルキルより選択される。いくつかの態様において、R^a、R^b、R^c、R^d、およびR^eは、任意で独立に、水素およびC_1-6アルキル、例えば、メチルからなる群より選択される。全ての不斉中心について、非対称基はR方向またはS方向のいずれかで見出され得、例えば、2種の例示的な立体化学的異性体には、以下のように図示され得るβ-Dアイソフォームおよびα-Lアイソフォームが含まれる。

In some embodiments, the LNA used in the oligonucleotide compounds of the invention preferably has a structure of general formula II:

Wherein Y is selected from the group consisting of —O—, —CH ₂ O—, —S—, —NH—, N (R ^e ), and / or —CH ₂ —;

Are independently selected from among internucleotide linkages, R ^H , terminal groups, or protecting groups; B constitutes a natural or non-natural nucleotide base moiety (nucleobase), R ^H is hydrogen and C _{1 -4} - is selected from ^{alkyl; R a, R b, R} c, R d, and R ^e are independently optionally, hydrogen, optionally substituted C _1-12 - alkyl, optionally substituted Good C _2-12 -alkenyl, optionally substituted C _2-12 -alkynyl, hydroxy, C _1-12 -alkoxy, C _{2-12 -alkoxyalkyl} , C _2-12 -alkenyloxy, carboxy, C _{1 -12} - alkoxycarbonyl, C _1-12 - alkyl, formyl, aryl, aryloxy - carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryl - aryloxy - carbonyl, heteroaryloxy, heteroarylcarbonyl, A Bruno, mono- - and di (C _1-6 - alkyl) amino, carbamoyl, mono- - and di (C _1-6 - alkyl) - amino - carbonyl, amino -C _1-6 - alkyl - aminocarbonyl, mono - and Di (C _1-6 -alkyl) amino-C _1-6 -alkyl-aminocarbonyl, C _1-6 -alkyl-carbonylamino, carbamide, C _1-6 -alkanoyloxy, sulphono, C _1-6 -Alkylsulfonyloxy, nitro, azide, sulfanyl, C _1-6 -alkylthio, halogen, DNA intercalator, group having photochemical activity, group having thermochemical activity, chelating group, reporter group, and ligand Wherein aryl and heteroaryl may be substituted, and the two geminal substituents R ^a and R ^b may both represent optionally substituted methylene (═CH ₂ ). ; R ^H , Hydrogen and C _1-4 - is selected from alkyl. In some embodiments, R ^a , R ^b , R ^c , R ^d , and ^Re are optionally independently selected from the group consisting of hydrogen and C _1-6 alkyl, eg, methyl. For all asymmetric centers, asymmetric groups can be found in either the R or S direction, for example, for two exemplary stereochemical isomers, β-D, which can be illustrated as follows: Isoforms and α-L isoforms are included.

Specific exemplary LNA units are shown below.

The term “thio-LNA” includes locked nucleotides wherein Y in the above general formula is selected from S or —CH ₂ —S—. Thio-LNA can be in both β-D and α-L configurations.

The term “amino-LNA” includes Y in the general formula above as —N (H) —, N (R) —, CH ₂ —N (H) —, and —CH ₂ —N (R) — ( Wherein R is selected from hydrogen and C _1-4 -alkyl). Amino-LNA can be in both β-D and α-L configurations.

  The term “oxy-LNA” includes locked nucleotides where Y in the general formula above represents —O—. Oxy-LNA can be in both β-D and α-L configurations.

In the term “ENA”, Y in the above general formula is —CH ₂ —O— (wherein the oxygen atom of —CH ₂ —O— is attached to the base 2 ′ position). Certain locked nucleotides are included. ^Re is hydrogen or methyl.

  In some exemplary embodiments, the LNA is selected from β-D-oxy-LNA, α-L-oxy-LNA, β-D-amino-LNA, and β-D-thio-LNA, specifically Is β-D-oxy-LNA.

RNAse recruitment It is recognized that oligomeric compounds can function through, for example, steric hindrance of translation or other methods through non-RNase-mediated degradation of target mRNA. In some embodiments, the oligomers of the invention can recruit endoribonucleases (RNases), eg, RNaseH.

  Such an oligomer, such as region A or a contiguous nucleotide sequence, preferably comprises a region of at least 6 contiguous nucleotide units, such as at least 7 contiguous nucleotide units, such as at least 8 or at least 9 contiguous nucleotide units (residues). 7 consecutive nucleotides, 8 consecutive nucleotides, 9 consecutive nucleotides, 10 consecutive nucleotides, 11 consecutive nucleotides, 12 consecutive nucleotides, 13 consecutive nucleotides, 14 consecutive nucleotides, 15 consecutive nucleotides, or 16 consecutive nucleotides, and with a complementary target RNA RNase can be recruited when a double strand is formed. The contiguous sequence that can recruit RNAse can be the region Y ′ referred to with respect to the gapmers described herein. In some embodiments, the size of a contiguous sequence that can recruit RNAse, e.g., region Y ', is larger, e.g., 10 nucleotide units, 11 nucleotide units, 12 nucleotide units, 13 nucleotide units, 14 nucleotide units, It may be 15 nucleotide units, 16 nucleotide units, 17 nucleotide units, 18 nucleotide units, 19 nucleotide units, or 20 nucleotide units.

  EP 1 222 309 provides an in vitro method for determining RNaseH activity that can be used to determine the ability to recruit RNaseH. When prepared with a complementary RNA target, using the methodology provided by Examples 91-95 of EP 1 222 309, it has the same base sequence but contains only DNA monomers, and 2 ′ At least 1% of the initial rate determined using a DNA-only oligonucleotide that has no substitution and has a phosphorothioate linking group between all monomers in the oligonucleotide, e.g., at least 5%, e.g., An oligomer is considered capable of recruiting RNaseH if it has an initial rate measured in pmol / l / min of at least 10%, or greater than 20%.

  In some embodiments, when prepared with a complementary RNA target and RNaseH, the RNaseH initial measured in pmol / l / min using the methodology provided by Examples 91-95 of EP 1 222 309. The rate is less than 1% of the initial rate determined using an equivalent DNA-only oligonucleotide having no 2 'substitution and having a phosphorothioate linking group between all nucleotides in the oligonucleotide, e.g. , Less than 5%, such as less than 10%, or less than 20%, the oligomer is considered essentially unable to recruit RNaseH.

  In other embodiments, the initial RNaseH rate measured in pmol / l / min using the methodology provided by Examples 91-95 of EP 1 222 309 when prepared with a complementary RNA target and RNaseH At least 20% of the initial rate determined using an equivalent DNA-only oligonucleotide that has no 2 'substitution and has a phosphorothioate linking group between all nucleotides in the oligonucleotide, e.g. An oligomer is considered capable of recruiting RNaseH if it is at least 40%, such as at least 60%, such as at least 80%.

  Typically, the region of the oligomer that forms a contiguous nucleotide unit capable of recruiting RNases when forming a duplex with a complementary target RNA forms a DNA / RNA-like duplex with the RNA target Consists of nucleotide units. The oligomers of the invention, for example the first region, may comprise a nucleotide sequence comprising both nucleotides and nucleotide analogues and may be in the form of gapmers, headmers, or mixmers, for example.

  A “headmer” is defined as an oligomer comprising a region X ′ and a continuous region Y ′ where the 5 ′ most monomer of region Y ′ is linked to the 3 ′ most monomer of region X ′. The Region X ′ includes a continuous stretch of nucleoside analogs that do not recruit RNase, and region Y ′ is a continuous stretch of DNA or nucleoside analog monomers that are recognizable and cleavable by RNase (eg, at least 7 consecutive monomers )including.

  A “tailmer” is an oligomer comprising a region X ′ and a continuous region Y ′ in which the 5 ′ monomer in region Y ′ is linked to the 3 ′ monomer in region X ′. Defined. Region X ′ contains a continuous stretch of DNA or nucleoside analog monomers that are recognizable and cleavable by RNase (eg, at least 7 consecutive monomers), and region X ′ is a continuous nucleoside analog that does not recruit RNase Including stretch.

  Other “chimeric” oligomers, referred to as “mixmers”, are alternating (i) DNA or nucleoside analog monomers that are recognizable and cleavable by RNases and (ii) nucleoside analog monomers that do not recruit RNases. Of the composition.

  In some embodiments, in addition to increasing the affinity of the oligomer for the target region, some nucleoside analogs also mediate RNase (eg, RNaseH) binding and cleavage. Since α-L-LNA (BNA) monomers recruit RNaseH activity to some extent, in some embodiments, oligomeric gap regions containing α-L-LNA monomers (eg, referred to herein) Region Y ′) consists of fewer monomers that are recognizable and cleavable by RNaseH, and introduces greater flexibility in the construction of the mixmer.

Gapmer Design In some embodiments, the oligomer of the invention, eg, the first region comprises or is a gapmer. A gapmer oligomer is a continuous stretch of nucleotides capable of recruiting RNAse, eg, RNAseH, eg, an oligomer comprising a region of at least 6 DNA nucleotides or 7 DNA nucleotides, referred to herein as region Y ′ (Y ′) And both regions 5 ′ and 3 ′ of region Y ′ are flanked by regions of affinity enhancing nucleotide analogues. For example, 1-6 nucleotide analogs are flanked by 5 'and 3' of a continuous stretch of nucleotides that can recruit RNAse. These regions are called a region X ′ (X ′) and a region Z ′ (Z ′), respectively. Examples of gapmers are disclosed in WO2004 / 046160, WO2008 / 113832, and WO2007 / 146511.

  In some embodiments, monomers capable of recruiting RNAse are DNA monomers, α-L-LNA monomers, C4 ′ alkylated DNA monomers (PCT / EP2009 / 050349 and Vester et al, incorporated herein by reference). Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300), and UNA (unlinked nucleic acid) nucleotides (Fluiter et al., Mol. Biosyst., 2009, 10 incorporated herein by reference). , 1039). UNA is typically an unlocked nucleic acid in which the C2-C3 C—C bond of ribose has been removed to form an unlocked “sugar” residue. Preferably, the gapmer comprises a (poly) nucleotide sequence of formula (5′-3 ′) X′-Y′-Z ′; region X ′ (X ′) (5 ′ region) comprises at least one A nucleotide analog, eg, consisting of or comprising at least one BNA (eg, LNA) unit, eg, 1-6 nucleotide analogues, eg, BNA (eg, LNA) unit; region Y ′ (Y ′ ) Consists of or comprises at least 5 contiguous nucleotides (eg, DNA nucleotides) that can recruit RNAse (when forming a duplex with a complementary RNA molecule, eg, mRNA target), And region Z ′ (Z ′) (3 ′ region) comprises at least one nucleotide analogue, such as at least one BNA (eg LNA unit), eg 1-6 nucleotide analogues, eg BNA Consists of or includes (eg, LNA) units.

  In some embodiments, region X ′ is one, two, three, four, five, or six nucleotide analogues, eg, BNA (eg, LNA) units, eg, 2-5 Nucleotide analogues, eg consisting of 2 to 5 LNA units, eg 3 or 4 nucleotide analogues, eg 3 or 4 LNA units; and / or region Z ′ is 1, 2 3, 4, 5, or 6 nucleotide analogues, eg BNA (eg LNA) units, eg 2-5 nucleotide analogues, eg 2-5 BNA (eg LNA units) ), Eg, 3 or 4 nucleotide analogues, eg 3 or 4 BNA (eg LNA) units.

  In some embodiments, Y ′ recruits 5, 6, 7, 8, 9, 10, 11, or 12 contiguous nucleotides, or RNAse, that can recruit RNAse. It can consist of or comprise 6-10 or 7-9, for example 8 consecutive nucleotides. In some embodiments, region Y ′ comprises at least one DNA nucleotide unit, such as 1 to 12 DNA units, preferably 4 to 12 DNA units, more preferably 6 to 10 DNA units. For example, consisting of or comprising 7 to 10 DNA units, most preferably 8, 9, or 10 DNA units.

  In some embodiments, region X ′ consists of 3 or 4 nucleotide analogs, such as BNA (eg, LNA), and region X ′ is 7, 8, 9, or 10 DNA. Composed of units and region Z ′ consists of 3 or 4 nucleotide analogues, eg BNA (eg LNA). Such designs include (X'-Y'-Z ') 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9 -3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3.

  Further gapmer designs are disclosed in WO2004 / 046160, which is incorporated herein by reference. WO 2008/113832 claiming priority based on US Provisional Application No. 60 / 977,409, which is incorporated herein by reference, refers to “shortmer” gapmer oligomers. In some embodiments, the oligomers presented herein may be such short mer gapmers.

  In some embodiments, the oligomer, eg, region X ′ comprises or is the formula (5′-3 ′) X′-Y′-Z ′, a total of 10 nucleotide units, 11 nucleotide units, It consists of a continuous nucleotide sequence of 12 nucleotide units, 13 nucleotide units, or 14 nucleotide units. X ′ consists of 1, 2 or 3 nucleotide analogue units, eg BNA (eg LNA) units; Y ′ is a duplex with a complementary RNA molecule (eg mRNA target) Consists of 7, 8, or 9 contiguous nucleotide units that can recruit RNAse when formed; and Z ′ is one, two, or three nucleotide analog units such as BNA ( For example, it consists of LNA) units.

  In some embodiments, X ′ consists of one BNA (eg, LNA) unit. In some embodiments, X ′ consists of two BNA (eg, LNA) units. In some embodiments, X ′ consists of 3 BNA (eg, LNA) units. In some embodiments, Z ′ consists of one BNA (eg, LNA) unit. In some embodiments, Z ′ consists of two BNA (eg, LNA) units. In some embodiments, Z ′ consists of 3 BNA (eg, LNA) units. In some embodiments, Y ′ consists of 7 nucleotide units. In some embodiments, Y ′ consists of 8 nucleotide units. In some embodiments, Y ′ consists of 9 nucleotide units. In certain embodiments, region Y ′ consists of 10 nucleoside monomers. In certain embodiments, region Y ′ consists of or comprises 1 to 10 DNA monomers. In some embodiments, Y ′ comprises 1 to 9 DNA units, eg, 2, 3, 4, 5, 6, 7, 8, or 9 DNA units. In some embodiments, Y ′ consists of DNA units. In some embodiments, Y ′ is at least one BNA unit in the α-L configuration, such as 2, 3, 4, 5, 6, 7, 8 in the α-L configuration. Includes 9 or 9 LNA units. In some embodiments, Y ′ comprises at least one α-L-oxy BNA / LNA unit, or all LNA units in the α-L configuration are α-L-oxy LNA units. In some embodiments, the number of nucleotides present in X′-Y′-Z ′ is selected from the group consisting of (nucleotide analog unit-region Y′-nucleotide analog unit): 1-8 -1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2 1-8-4, 2-8-4, or 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2 , 1-9-3, 3-9-1, 4-9-1, 1-9-4, or 1-10-1, 1-10-2, 2-10-1, 2-10-2 , 1-10-3, 3-10-1. In some embodiments, the number of nucleotides in X′-Y′-Z ′ is 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3 , 3-7-2, 3-7-4, and 4-7-3. In certain embodiments, each of regions X ′ and Y ′ consists of 3 BNA (eg, LNA) monomers, and region Y ′ is 8 or 9 or 10 nucleoside monomers, preferably DNA Consists of monomers. In some embodiments, both X ′ and Z ′ are each composed of 2 BNA (eg, LNA) units and Y ′ is composed of 8 or 9 nucleotide units, preferably DNA units. In various embodiments, other gapmer designs include regions X ′ and / or Z ′ where 3, 4, 5, or 6 nucleoside analogs, such as 2′-O-methoxyethyl- A monomer containing a ribose sugar (2′-MOE) or a monomer containing a 2′-fluoro-deoxyribose sugar), and the region Y ′ is 8, 9, 10, 11, Or consisting of 12 nucleosides, for example DNA monomers, where the region X′-Y′-Z ′ is 3-9-3, 3-10-3, 5-10-5, or 4-12 Includes those having 4 monomers. Further gapmer designs are disclosed in WO2007 / 146511A2, which is incorporated herein by reference.

Splice switching oligomers In some embodiments, the antisense oligonucleotide is a splice switching oligomer, ie, an oligomer that targets pre-mRNA that causes alternative splicing of the pre-mRNA.

  Splice switching oligomer targets may include TNF receptors, for example, splice switching oligomers (SSO) are disclosed in WO2007 / 058894, WO08051306 A1, and PCT / EP2007 / 061211, which are incorporated herein by reference. There may be one or more of TNFR SSO.

  Splice switching oligomers are typically (essentially) unable to recruit RNaseH, and therefore gapmer, tailmer, or headmer designs are generally undesirable. However, the mixmer design and totalmer design are suitable for SSO.

  Splice switching oligomers have also been used to target dystrophin deficiency in Duchenne muscular dystrophy.

Mixmers Most antisense oligonucleotides are compounds that are designed to recruit RNase enzymes (eg, RNaseH) to degrade their intended target. Such compounds include DNA phosphorothioate oligonucleotides, as well as gapmers, headmers, and tailmers. These compounds typically comprise a region of at least 5 or 6 DNA nucleotides, and in the case of gapmers, affinity enhancing nucleotide analogues are flanked on both sides. The oligomers of the present invention may operate via an RNase (eg, RNaseH) independent mechanism. Examples of oligomers that operate via non-RNase H (or non-RNase) mechanisms are mixmers and totalmers.

  The term “mixmer” is in contrast to gapmers, tailmers, and headmers, where there are no more than 5 flanking sequences, and in some embodiments, more than 4, for example, more than 3 consecutive Naturally occurring nucleotides, such as oligomers that contain both naturally occurring and non-naturally occurring nucleotides in the absence of DNA units. In some embodiments, the mixmer does not comprise more than 5 consecutive nucleoside analogs, eg, BNA (LNA), and in some embodiments, more than 4, eg, more than 3 consecutive nucleoside analogs. For example, BNA (LNA) is not included. In such a mixmer, the remaining nucleosides can be, for example, DNA nucleosides, and / or non-bicyclic nucleoside analogs, such as those referred to herein, such as 2′-substituted nucleoside analogs, such as , 2′-O-MOE or 2 ′ fluoro.

  The oligomer according to the invention can be a mixmer, indeed as an oligomer or its first region (especially when targeting a microRNA (anti-miR), a microRNA binding site (Blockmir) on mRNA) or a splice. As a switching oligomer (SSO), various mixmer designs are highly effective. See, for example, WO2007 / 112754 (LNA-anti-miR ™) and WO2008 / 131807 (LNA splice switching oligo).

  In some embodiments, the oligomer or mixmer may optionally include BNA and 2 ′ substituted nucleoside analogs along with DNA nucleosides. See, for example, WO07027894 and WO2007 / 112754, which are incorporated herein by reference. Specific examples include LNA and 2'-O-MOE and DNA, LNA and 2'fluoro and 2'-O-MOE, 2'-O-MOE and 2'fluoro, 2'-O-MOE and 2'fluoro And LNA, or an oligomer or first region comprising LNA, 2′-O-MOE, LNA and DNA.

  In some embodiments, the oligomer or mixmer comprises or consists of a nucleotide sequence and a naturally occurring nucleotide, or a contiguous nucleotide sequence of a repeating pattern of one type of nucleotide analog and a second type of nucleotide analog. Become. The repetitive pattern is, for example, every 2 or 3 nucleotides are nucleotide analogues such as BNA (LNA) and the remaining nucleotides are naturally occurring nucleotides such as DNA or 2 'Substituted nucleotide analogs, such as 2'MOE analogs or 2' fluoro analogs referred to herein, or in some embodiments, of the nucleotide analogs referred to herein. It can be selected from a group. It will be appreciated that a repeating pattern of nucleotide analogs, eg, LNA units, may be combined with nucleotide analogs at fixed positions, eg, 5 ′ or 3 ′ ends.

  In some embodiments, the first nucleotide of the oligomer or mixmer, counting from the 3 ′ end, is a nucleotide analog, eg, an LNA nucleotide.

  In some embodiments that may be the same or different, the second nucleotide of the oligomer or mixmer, counting from the 3 ′ end, is a nucleotide analog, eg, an LNA nucleotide.

  In some embodiments, which may be the same or different, the 7th and / or 8th nucleotides of the oligomer or mixmer, counting from the 3 ′ end, are nucleotide analogues, such as LNA nucleotides. It is.

  In some embodiments, which may be the same or different, the 9th and / or 10th nucleotides of the first oligomer and / or the second oligomer, counted from the 3 ′ end, are nucleotide analogues For example, LNA nucleotides.

  In some embodiments, which may be the same or different, the 5 ′ end of the oligomer or mixmer is a nucleotide analog, eg, an LNA nucleotide.

  The design features described above can be incorporated into mixmer designs, such as anti-miR mixmers, in some embodiments.

  In some embodiments, the oligomer or mixmer does not comprise a region of more than 4 consecutive DNA nucleotide units or more than 3 consecutive DNA nucleotide units. In some embodiments, the mixmer does not comprise a region of more than two contiguous DNA nucleotide units.

  In some embodiments, the oligomer or mixmer comprises at least a region consisting of at least two consecutive nucleotide analog units, eg, at least two consecutive LNA units.

  In some embodiments, the oligomer or mixmer comprises at least a region consisting of at least 3 contiguous nucleotide analog units, eg, at least 3 contiguous LNA units.

  In some embodiments, the oligomers or mixmers of the invention do not comprise a region of more than 7 consecutive nucleotide analog units, eg, LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 6 consecutive nucleotide analog units, eg, LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 5 consecutive nucleotide analog units, eg, LNA units. In some embodiments, the oligomer or mixmer of the present invention does not comprise a region of more than 4 consecutive nucleotide analog units, eg, LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than 3 consecutive nucleotide analog units, eg, LNA units. In some embodiments, the oligomer or mixmer of the invention does not comprise a region of more than two consecutive nucleotide analog units, eg, LNA units. A mixmer is an oligomer that can contain one or more short regions of DNA of no more than four consecutive DNA nucleotides, typically alternating regions of nucleotide analogs (eg, LNA units) and DNA nucleotides, optionally , Including regions of other nucleotide analogs (eg, non-LNA nucleotide analogs). Totalmers do not contain DNA or RNA nucleotides (although they can contain analogs or derivatives of DNA and RNA). In some embodiments, the oligomer of the invention (eg, region A) may comprise, in some embodiments, no more than 4 contiguous DNA nucleotides or no more than 3 contiguous DNA nucleotides.

  The following embodiments may apply to mixmer or totalmer oligomers (eg, as region A): oligomers of the invention (eg, region A) may in some embodiments be LNA nucleotides and non-LNA nucleotides (eg, At least two alternating regions of DNA or 2 ′ substituted nucleotide analogues).

The oligomers of the invention may comprise, in some embodiments, a contiguous sequence of the formula: 5 ′ ([LNA nucleotides] _1-5 and [non-LNA nucleotides] _1-4 ) _2-12.3 ′.

  In some embodiments, the 5 ′ nucleotide of the contiguous nucleotide sequence (or oligomer) is an LNA nucleotide.

  In some embodiments, the 3 ′ nucleotide of the contiguous nucleotide sequence is a nucleotide analog, such as LNA, or 2, 3, 4, 5 3 ′ nucleotides are nucleotide analogs, such as LNA. Nucleotides, or other nucleotide analogs that confer enhanced serum stability to oligomers.

In some embodiments, the continuous nucleotide sequence of the oligomer has the formula 5 ′ ([LNA nucleotides] _1-5- [non-LNA nucleotides] _1-4 ) _2-11- [LNA nucleotides] _1-5 3 ′.

In some embodiments, the contiguous nucleotide sequence of the oligomer has 2, 3, or 4 contiguous regions of LNA nucleotides and non-LNA nucleotides, for example, the formula 5 ′ ([LNA nucleotides] _1-5 and [ Non-LNA nucleotides] _1-4 ) _2-3 and optionally further 3'LNA regions [LNA nucleotides] _1-5 .

In some embodiments, the contiguous nucleotide sequence of the oligomer comprises 5 ′ ([LNA nucleotides] _1-3 and [non-LNA nucleotides] _1-3 ) _2-5 , optionally with additional 3 ′ LNA regions [LNA nucleotides ] Including _1-3 .

In some embodiments, the contiguous nucleotide sequence of the oligomer comprises 5 ′ ([LNA nucleotides] _1-3 and [non-LNA nucleotides] _1-3 ) ₃ , optionally with an additional _{3 ′} LNA region [LNA nucleotide] ₁ Includes _-3 .

  In some embodiments, all non-LNA nucleotides are DNA nucleotides.

  In some embodiments, the non-LNA nucleotide is a DNA unit, RNA unit, 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, and 2′-fluoro-DNA. Selected independently or dependently from the group of units.

  In some embodiments, the non-LNA nucleotide is (optionally independently) selected from the group consisting of 2′-substituted nucleoside analogs, eg, (optionally independently) 2′-O-alkyl-RNA units, 2 ′ -OMe-RNA units, 2'-amino-DNA units, 2'-AP, 2'-FANA, 2 '-(3-hydroxy) propyl, and 2'-fluoro-DNA units, and / or other (optional) From the group consisting of saccharide-modified nucleoside analogues, eg, morpholinos, peptide nucleic acids (PNA), CeNA, unlinked nucleic acids (UNA), hexitol nucleic acids (HNA), bicyclo-HNA (eg see WO2009 / 100320) Selected. In some embodiments, the nucleoside analog increases the affinity of the first region for its target nucleic acid (or complementary DNA or RNA sequence). Various nucleoside analogs are described by Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3 (2), 293, incorporated herein by reference. -213.

  In some embodiments, the non-LNA nucleotide is a DNA nucleotide. In some embodiments, the oligomer or continuous nucleotide sequence comprises LNA nucleotides, and optionally other nucleotide analogs (eg, non-LNA) that may be affinity enhancing nucleotide analogs and / or serum stability enhancing nucleotide analogs. Nucleotide analogues listed for nucleotides).

  In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of the contiguous nucleotide sequence of the nucleotide analog.

  In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of a contiguous nucleotide sequence of LNA nucleotides.

  In some embodiments, the oligomer or continuous nucleotide sequence is 8-12 nt long, such as 8-10 nt long or 10-20 nt long, such as 12-18 nt long or 14-16 nt long.

In some embodiments, the oligomer or contiguous nucleotide sequence comprises a duplex having a T _{m of} at least about 60 ° C., for example, at least 65 ° C., with a complementary single-stranded RNA nucleic acid molecule having a phosphodiester internucleoside linkage. Can be formed.

Example of T _m assay: oligonucleotide: oligonucleotide and RNA target (PO) duplex diluted to 3 mM with 500 ml RNase-free water and 500 ml 2 × T _m buffer (200 mM NaCl, 0.2 mM EDTA) , 20 mM Na phosphate, pH 7.0). The solution is heated to 95 ° C. for 3 minutes and then annealed at room temperature for 30 minutes. The duplex melting temperature (T _m ) is measured with a Lambda 40 UV / VIS spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is raised from 20 ° C. to 95 ° C. and then lowered to 25 ° C. and the absorbance at 260 nm is recorded. The first derivative and maxima of both melting and annealing, is used to assess duplex T _m.

Totalmers Totalmers are single-stranded oligomers that contain only non-naturally occurring nucleosides, eg, sugar-modified nucleoside analogs.

  The first region according to the present invention may be a totalmer, indeed, as an oligomer or its first region (eg, especially when targeting microRNA (anti-miR)), or as a splice switching oligomer (SSO) Totalmer design is highly effective. In some embodiments, the totalmer has at least one XYX or YXY sequence motif, such as the sequence XYX or YXY (wherein X is an LNA and Y is an alternative (ie, non-LNA ) Containing or consisting of repeats of nucleotide analogues, such as 2′-O-MOE RNA units and 2′-fluoro DNA units. The sequence motif can be, for example, XXY, XYX, YXY, or YYX in some embodiments.

  In some embodiments, the totalmer comprises a continuous nucleotide sequence of 7-16 nucleotides, such as 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, or 15 nucleotides, such as 7-12 nucleotides. Can contain or consist of.

  In some embodiments, the contiguous nucleotide sequence of the totalmer is at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as At least 90%, eg, 95%, eg, 100%, containing BNA (LNA) units The remaining units may be selected from the non-LNA nucleotide analogs referred to herein, such as 2′-O_alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, Selected from the group consisting of 2'-fluoro-DNA units, LNA units, PNA units, HNA units, INA units, and 2'MOE RNA units, or the group consisting of 2'-OMe RNA units and 2'-fluoro DNA units obtain.

  In some embodiments, the totalmer consists of or comprises a contiguous nucleotide sequence consisting only of LNA units. In some embodiments, a totalmer, eg, an LNA totalmer, is 7-12 nucleoside units long. In some embodiments, the totalmer (as an oligomer or as its first region) can be targeted to microRNA (ie, as mentioned in WO2009 / 043353, which is incorporated herein by reference) Anti-miR).

  In some embodiments, the oligomer or continuous nucleotide sequence comprises LNA nucleotides and optionally other nucleotide analogues that can be affinity enhancing nucleotide analogues and / or serum stability enhancing nucleotide analogues.

  In some embodiments, the oligomer or contiguous nucleotide sequence thereof consists of the contiguous nucleotide sequence of the nucleotide analog.

  In some embodiments, the oligomers or their contiguous nucleotide sequences consist of contiguous nucleotide sequences of LNA nucleotides.

Modulation of microRNA through an oligomer or its first region In some embodiments, the oligomer or its first region comprises or is a contiguous nucleotide sequence that corresponds to or is completely complementary to the entire mature microRNA. Is anti-miR. The use of the present invention to modulate the in vivo activity of microRNAs is considered to be most important due to the fact that microRNAs typically control a large number of mRNAs in a subject. Thus, the ability to inactivate therapeutic anti-miRs is highly desirable.

A number of microRNAs are associated with a number of diseases. For example, non-limiting examples of therapeutic indications that can be treated with the pharmaceutical composition of the invention are the following.

  The tumor suppressor gene tropomysin 1 (TPM1) mRNA has been shown as a target for miR-21. Myotrophin (mtpn) mRNA has been shown as a target for miR375. Thus, the oligomer or its first region targets one of the microRNAs listed above (ie comprises or consists of a contiguous nucleotide sequence that is completely complementary to its corresponding region). Or an anti-mir that contains no more than one mismatch to it.

  Accordingly, some aspects of the invention include infectious diseases such as viral diseases such as hepatitis C virus and HIV, fragile X mental retardation, inflammatory diseases, cancers such as chronic lymphocytic leukemia, breast cancer Relates to the treatment of diseases associated with the expression of microRNAs selected from the group consisting of lung cancer and colon cancer.

  MicroRNAs (miRNAs) are a rich class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base pairing with target mRNAs. Mature miRNAs are sequentially processed from longer hairpin transcripts by the RNAse III ribonuclease draw. Mature microRNA (miR) typically contains 20-25 contiguous RNA nucleotides. Several microRNAs are now widely established to be associated with medical conditions and diseases, and several companies mimic microRNAs or are specific for disease phenotypes. An oligomer-based therapeutic that is specifically hybridized with microRNA is under development. Such oligomers are referred to herein as microRNA mimics and anti-miR, respectively, and the oligomer or its first region may in some embodiments be an oligomer that modulates such microRNA. .

  In some embodiments, the oligomer according to the invention or the first region thereof is a microRNA sequence, such as a mature microRNA sequence, such as miRBase (http://microrna.sanger.ac.uk/cgi-bin/sequences/ mirna_summary.pl?org=hsa) consisting of or containing a contiguous nucleotide sequence corresponding to or completely complementary to human microRNA. In some embodiments, the microRNA is a viral microRNA. At the time of this writing, miRbase 19 has 1600 precursors and 2042 mature human miRNA sequences within miRBase, all referenced, including the mature microRNA sequence of each human microRNA. Is incorporated herein by reference. Other human microRNAs that can be targeted by the oligomer or its first region include those disclosed in WO08040355A, which is incorporated herein by reference. In some embodiments, the oligomer according to the invention or the first region thereof is from the group consisting of hsa-miR19b, hsa-miR21, hsa-miR 122, hsa-miR 142 a7b, hsa-miR 155, and hsa-miR 375. Consists of or includes a contiguous nucleotide sequence that corresponds to or is completely complementary to the selected microRNA sequence. In some embodiments, the oligomer according to the invention or the first region thereof consists of a contiguous nucleotide sequence corresponding to or completely complementary to a microRNA sequence selected from the group consisting of hsa-miR221 and hsa-miR222 Or including it. In some embodiments, the oligomer according to the invention or the first region thereof is from a contiguous nucleotide sequence that corresponds to or is completely complementary to hsa-miR122 (NR — 029667.1 GI: 262205241), eg, mature has-miR122. Become or include it.

  In some embodiments, when the oligomer or its first region targets miR-122, the oligomer is for use in the treatment of hepatitis C infection.

Anti-miR oligomers Preferred oligomers or “anti-miR” designs and oligomers of the first region are described in WO2007 / 112754, WO2007 / 112753, PCT / DK2008 / 000344, and US provisional applications 60/979217 and 61/028062. Which are all incorporated herein by reference. In some embodiments, the oligomer or first region thereof is an anti-miR that is a mixmer or a totalmer.

  An anti-miR oligomer is a contiguous nucleotide sequence that is completely complementary or essentially complementary (ie, may contain one or two mismatches) to the microRNA sequence or its corresponding subsequence. An oligomer consisting of or containing it. In this regard, the anti-miR may comprise a contiguous nucleotide sequence that is complementary or essentially complementary to the entire mature microRNA, or the anti-miR is a mature microRNA or pre-microRNA. It is contemplated that it may include a contiguous nucleotide sequence that is complementary or essentially complementary to a subsequence, and such a subsequence (and thus the corresponding contiguous nucleotide sequence) is typically At least 8 nucleotides, e.g., 8-25 nucleotides, e.g., 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides Plastid length, 24 nucleotides in length, e.g., 10 to 17 nucleotides or 10 to 16 nucleotides, for example, 12 to 15 nucleotides.

  Numerous designs of anti-miR have been proposed, and typically the anti-miR for therapeutic use, eg, its contiguous nucleotide sequence, contains one or more nucleotide analog units.

  In some embodiments, the anti-miR can have a gapmer structure as described herein. However, as described in WO2007 / 112754 and WO2007 / 112753, other designs such as mixmers or totalmers may also be preferred.

  WO2007 / 112754 and WO2007 / 112753, both incorporated herein by reference, provide anti-miR oligomers and anti-miR oligomer designs that are complementary to mature microRNAs.

  In some embodiments, the anti-miR subsequence corresponds to a miRNA seed region. In some embodiments, the first or second 3 ′ nucleobase of the oligomer corresponds to the second 5 ′ nucleotide of the microRNA sequence.

  In some anti-miR embodiments, oligomeric nucleobase units 1-6 (including both ends) measured from the 3 ′ end of the oligomeric region are complementary to the microRNA seed region sequence.

  In some anti-miR embodiments, oligomeric nucleobase units 1-7 (including both ends) measured from the 3 ′ end of the oligomeric region are complementary to the microRNA seed region sequence.

  In some anti-miR embodiments, oligomeric nucleobase units 2-7 (including both ends) measured from the 3 ′ end of the oligomeric region are complementary to the microRNA seed region sequence.

  In some embodiments, the anti-miR oligomer comprises at least one nucleotide analog unit, eg, at least one LNA unit, at a position in a region complementary to the miRNA seed region. The anti-miR oligomer, in some embodiments, is 1-6 or 1-7 nucleotide analog units, such as 1-6 and 1-7, in positions complementary to the miRNA seed region. Of LNA units.

  In some embodiments, an anti-miR of the invention is 7 nucleotides, 8 nucleotides, or 9 nucleotides long, comprises a contiguous nucleotide sequence complementary to a seed region of a human or viral microRNA, and comprises at least nucleotides 80%, such as 85%, such as 90%, such as 95%, such as 100%, is LNA.

  In some embodiments, an anti-miR of the invention is 7 nucleotides, 8 nucleotides, or 9 nucleotides long and comprises a contiguous nucleotide sequence that is complementary to the seed region of a human or viral microRNA, and comprises at least nucleotides 80% is LNA and at least 80%, such as 85%, such as 90%, such as 95%, such as 100% of the internucleotide linkages are phosphorothioate linkages.

  In some embodiments, the anti-miR comprises 1 or 2 LNA units at positions 3-8, counting from the 3 ′ end. This is thought to be advantageous for the stability of the A helix formed by the oligonucleotide: microRNA duplex, which is a duplex that is similar in structure to the RNA: RNA duplex.

  The table from page 48 line 15 to page 51 line 9 of WO2007 / 112754 provides an example of an anti-microRNA oligomer (ie, an anti-miR that may be an oligomer or a first region thereof) and is incorporated herein by reference. Specifically incorporated into the specification.

MicroRNA Mimics In some embodiments, the oligomer or first region thereof is in the form of an miRNA mimic that can be introduced into a cell to suppress expression of one or more mRNA targets. miRNA mimics are typically completely complementary to full-length miRNA sequences. An miRNA mimetic is a compound that comprises a contiguous nucleotide sequence that is homologous to a corresponding region of one or more of the miRNA sequences provided or referenced herein. The use of miRNA mimics or anti-miRs (optionally) further suppresses the mRNA target, or silences (down-regulates) the miRNA, thereby inhibiting the function of the endogenous miRNA and derepressing the mRNA target Can be used to cause and increase expression.

Aptamers In some embodiments, the oligomer or first region thereof can be a therapeutic aptamer, a spiegelmer. Note that aptamers may be ligands, eg, receptor ligands, and thus can be used as targeting moieties (ie, region 3). Aptamers (also called Spiegelmers) according to the present invention are 20-50 nucleotide long nucleic acids selected based on conformation rather than nucleotide sequence and have therapeutic effects by binding directly to the target protein in vivo. And therefore does not contain the reverse complementary strand of the target. In fact, their targets are proteins, not nucleic acids. Specific aptamers that can be oligomers or the first region thereof include Macugen (OSI Pharmaceuticals) or ARC1779 (Archemix, Cambridge, MA). In some embodiments, the oligomer or first region thereof is not an aptamer. In some embodiments, the oligomer or first region thereof is not an aptamer or a spiegelmer.

siRNA complexes In some embodiments, the oligomer or first region thereof can be part of an siRNA complex, ie, the antisense strand or passenger strand of the siRNA complex. siRNA complexes can mediate RNA interference.

  In some embodiments, the siRNA complex is 17-25 nt long, e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, e.g., 21- Contains two single-stranded oligomers 23 nucleotides long. In some embodiments, the sense and / or antisense strand of the siRNA can typically comprise a 1 nucleotide, 2 nucleotide, or 3 nucleotide 3 ′ overhang. Where appropriate, the sense and / or antisense strand may comprise one or more nucleotide analogues.

  In some embodiments, the siRNA complex is an siLNA, eg, the siRNA design described in WO2004 / 000192, WO2005 / 073378, WO2007 / 085485, all incorporated herein by reference. siLNA is an siRNA containing at least one LNA unit.

  In some embodiments, the siRNA complex is a sisiLNA, eg, those described in WO2007 / 107162, incorporated herein by reference. In some embodiments, the oligomer of the invention or the first region thereof is the sense strand of the siRNA and can therefore be non-complementary to the target (in fact, can be homologous to the intended target).

  In some embodiments, the oligomer or compound of the invention is not an siRNA or siLNA.

Internucleotide linkage The nucleoside monomers of the oligomers described herein (eg, the first region and the second region) are linked together via an [internucleoside] linking group. Optionally, each monomer is linked to the 3 ′ adjacent monomer via a linking group.

  In the context of the present invention, those skilled in the art will appreciate that the terminal 5 ′ monomer of the oligomer may or may not include a 5 ′ end group, but does not include a 5 ′ linking group. I will.

  The term “linking group” or “internucleotide linkage” refers to a group capable of covalently joining two nucleotides together. Specific preferred examples include phosphate groups and phosphorothioate groups.

  The oligomeric nucleotides of the invention or their contiguous nucleotide sequences are linked together through a linking group. Optionally, each nucleotide is linked to the 3 ′ adjacent nucleotide via a linking group.

  Suitable internucleotide linkages include those listed in WO2007 / 031091, for example those listed in the first paragraph on page 34 of WO2007 / 031091 (incorporated herein by reference).

  In some embodiments, it is preferred to modify phosphodiester linkages or internucleotide linkages other than region B from normal phosphodiesters to those that are more resistant to attack by nucleases, such as phosphorothioates or boranophosphates. . These two species can be cleaved by RNaseH and also provide a pathway for antisense inhibition in reducing target gene expression.

  Suitable sulfur (S) containing internucleotide linkages provided herein, such as phosphorothioates or phosphodithioates, may be preferred. Phosphorothioate internucleotide linkages are particularly preferred for the first region, for example, in gapmers, mixmers, anti-mirs, splice switching oligomers, and totalmers.

  For gapmers, the internucleotide linkage within the oligomer can be, for example, phosphorothioate or boranophosphate to allow cleavage of the targeted RNA by RNaseH. Phosphorothioates are preferred for improved nuclease resistance and other reasons, such as ease of manufacture.

  In one aspect, except for the phosphodiester linkage between the first region and the second region, and optionally excluding the phosphodiester linkage in region B, the remaining internucleoside linkage of the oligomer of the invention, nucleotides and / or Alternatively, nucleotide analogs are linked to each other by phosphorothioate groups. In some embodiments, at least 50%, eg, at least 70%, eg, at least 80%, eg, at least 90%, eg, all internucleoside linkages between nucleosides in the first region are phosphodiester (phosphate). For example, selected from the group consisting of phosphorothioate, phosphodithioate, or boranophosphate. In some embodiments, at least 50%, such as at least 70%, such as at least 80%, such as at least 90%, such as all internucleoside linkages between nucleosides in the first region are phosphorothioate.

  WO09124238 refers to oligomeric compounds having at least one bicyclic nucleoside attached to the 3 ′ or 5 ′ end by a neutral internucleoside linkage. Thus, the oligomers of the present invention can be 3′-terminal or 5 ′ by neutral internucleoside linkages such as one or more phosphotriesters, methylphosphonates, MMI, amide-3, formacetal, or thioformacetal. It may have at least one bicyclic nucleoside attached to the end. The remaining linkage can be phosphorothioate.

Conjugates, targeting moieties, and blocking groups The term “conjugate” refers to the covalent attachment (“conjugation”) of an oligomer described herein to one or more non-nucleotide or non-polynucleotide moieties. )) Is a heterogeneous molecule formed. Examples of non-nucleotide or non-polynucleotide moieties include macromolecular drugs such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically, the protein can be an antibody against the target protein. A typical polymer may be polyethylene glycol.

  Thus, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region, typically consisting of a contiguous sequence of nucleotides, and an additional non-nucleotide region. When referring to an oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise a non-nucleotide component, such as a conjugate component.

  In various embodiments of the invention, the oligomeric compound is linked to a ligand / conjugate that can be used, for example, to increase cellular uptake of the oligomeric compound. WO2007 / 031091 provides suitable ligands and conjugates, which are incorporated herein by reference.

  In various embodiments, where the compound of the invention consists of the specified nucleic acid or nucleotide sequence disclosed herein, the compound comprises at least one non-nucleotide moiety or non-polynucleotide covalently attached to the compound. A moiety (eg, not including one or more nucleotides or nucleotide analogs) may be included.

  In some embodiments, the conjugate is a lipophilic conjugate or protein (eg, antibody, enzyme, serum protein); peptide; vitamin (water soluble or fat soluble); polymer (water soluble or fat soluble); drug, toxin , Reporter molecules, and small molecules including receptor ligands; carbohydrate complexes; nucleic acid cleavage complexes; metal chelators (eg, porphyrins, texaphyrins, crown ethers, etc.); intercalators including hybrid photonucleases / intercalators Crosslinkers (eg, photoactive, redox activity), and combinations and derivatives thereof; A number of suitable conjugate moieties, their preparation and linkage with oligomeric compounds are provided, for example, in WO 93/07883 and US Pat. No. 6,395,492, each incorporated herein by reference in its entirety. Oligonucleotide conjugates and their synthesis are described in Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch. 16, Marcel Dekker, Inc., each of which is incorporated herein by reference in its entirety. , 2001, and in a comprehensive review by Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

  Conjugation (with the conjugate moiety) can enhance the activity, cell distribution, or cellular uptake of the oligomers of the invention. Such moieties include antibodies, polypeptides, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-s-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol residues or undecyl residues. Groups, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, polyamine chains or polyethylene glycol chains, adamantaneacetic acid, palmityl moieties, These include, but are not limited to, octadecylamine or hexylamino-carbonyl-oxacholesterol moieties.

  The oligomers of the invention may be conjugated to active drug substances such as aspirin, ibuprofen, sulfa drugs, antidiabetic drugs, antibacterial drugs, or antibiotics.

  In certain embodiments, the conjugated moiety is a sterol, such as cholesterol.

  In various embodiments, the conjugated moiety is a positively charged polymer, e.g., 1-50 amino acid residues long, e.g., 2-20 amino acid residues long, e.g., 3-10 amino acid residues long positive And / or polyalkylene oxides such as polyethylene glycol (PEG) or polypropylene ethylene glycol (see WO2008 / 034123, incorporated herein by reference).

  The use of conjugates is often associated with enhanced pharmacokinetic or pharmacodynamic dynamic properties. However, if the presence of a conjugate group interferes with the activity of the oligonucleotide against its intended target, for example via steric hindrance that prevents hybridization or nuclease recruitment (eg RNAseH or RISC recruitment) There is. The use of a DNA and / or RNA phosphodiester region (region B) between the oligonucleotide (region A) and the conjugate moiety (X) according to the invention allows for improved properties due to the presence of the conjugate group. In the target tissue, ensure that the conjugate group does not prevent the effective activity of the oligonucleotide.

  The oligonucleotides of the invention are in some embodiments covalently attached to one or more conjugate groups, optionally through one or more linkers. The resulting conjugated compound may have improved enhanced properties, eg, improved or enhanced pharmacokinetic properties, pharmacodynamic properties, compared to, for example, unconjugated oligomeric compounds, And may have other properties. Conjugate moieties that can improve or enhance the pharmacokinetic properties of an oligomeric compound improve the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligomeric compound. obtain. Conjugate moieties that can improve or enhance the pharmacodynamic properties of oligomeric compounds can improve activity, resistance to degradation, sequence-specific hybridization, uptake, and the like. In some embodiments, the conjugate group can reduce or prevent inappropriate activity of the oligonucleotide, eg, off-target activity, or activity in a non-target tissue or organ. This can be achieved, for example, by the use of a blocking moiety, which can be a conjugate, where the presence of a blocking group covalently attached to the oligonucleotide (optionally via a linker) can cause hybridization and / or activity of the oligonucleotide. Can prevent or block. Cleavage of the DNA / RNA phosphodiester region (eg, at the intended target site) removes the blocking group and allows delivery of the active oligonucleotide to the intended site.

  In some embodiments, the compounds of the invention include conjugate groups. For example, one conjugate group is used for targeting to a specific tissue, for example, a lipophilic group is used for targeting to the liver, to provide additional benefits, the second It will be appreciated that conjugate groups such as blocking groups or additional therapeutic entities may be used. Appropriate one or both of the conjugates / moieties can be linked to the oligonucleotide via the DNA / RNA phosphodiester region according to the present invention. In some embodiments, the conjugate is covalently attached to the oligonucleotide at the 5 ′ end and / or 3 ′ end of the oligonucleotide, optionally via a linker. In this regard, if two conjugates / subgroups are used, one can be linked to the 5 ′ end and one to the 3 ′ end.

Carbohydrate conjugates In some embodiments, the conjugate group is from the group consisting of carbohydrates, lipophilic moieties, polymers, proteins or peptides, labels or dyes, small molecules such as small molecule therapeutic moieties, cell surface receptor ligands. Selected.

  In some embodiments, the conjugate may be a carbohydrate or may include a carbohydrate or include a carbohydrate group. In some embodiments, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate. In some embodiments, the conjugate group may be or include mannose or mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in a range of tissues, eg, liver and / or muscle. See, for example, EP 1495769, WO99 / 65925, Yang et al., Bioconjug Chem (2009) 20 (2): 213-21, Zatsepin & Oretskaya Chem Biodivers. (2004) 1 (10): 1401-17.

  In some embodiments, the conjugate group is a carbohydrate moiety. Further, the oligomer may further comprise one or more additional conjugate moieties, of which lipophilic or hydrophobic moieties are of particular interest. These can act, for example, as pharmacokinetic modulators, and optionally a carbohydrate conjugate, a linker that links the carbohydrate conjugate to the oligomer, or a plurality of carbohydrate conjugates via a biocleavable linker. The (multivalent conjugate) can be linked to a linker that links the oligomer or covalently.

  In some embodiments, the conjugate may be a carbohydrate or may include a carbohydrate or include a carbohydrate group. In some embodiments, the carbohydrate is selected from the group consisting of galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate. In some embodiments, the conjugate group may be or include mannose or mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in a range of tissues, eg, liver and / or muscle. See, for example, EP 1495769, WO99 / 65925, Yang et al., Bioconjug Chem (2009) 20 (2): 213-21, Zatsepin & Oretskaya Chem Biodivers. (2004) 1 (10): 1401-17.

GalNAc conjugates The present invention also provides oligonucleotides, eg, LNA antisense oligomers, conjugated to asialoglycoprotein receptor targeting moieties. In some embodiments, the conjugate moiety (eg, third region or region C) is an asialoglycoprotein receptor targeting moiety, eg, galactose, galactosamine, N-formyl-galactosamine, N acetylgalactosamine, N-propionyl-galactosamine Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. In some embodiments, the conjugate comprises a galactose cluster, eg, N-acetylgalactosamine trimer. In some embodiments, the conjugate moiety comprises GalNAc (N-acetylgalactosamine), eg, monovalent, divalent, trivalent, or tetravalent GalNAc. Trivalent GalNAc conjugates can be used to target compounds to the liver. GalNAc conjugates include methylphosphonate and PNA antisense oligonucleotides (eg, US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov-Dec; 6 (6): 695-701) and siRNA (eg, WO2009 / 126933, WO2012 / 089352 & WO2012 / 083046). References to GalNAc and the specific conjugates used therein are incorporated herein by reference. WO2012 / 083046 discloses siRNAs having a GalNAc conjugate moiety that includes a cleavable pharmacokinetic modulator that is suitable for use in the present invention, wherein a preferred pharmacokinetic modulator is a C16 hydrophobic group such as palmitoyl, Hexadec-8-enoyl, oleyl, (9E, 12E) -octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The '046 cleavable pharmacokinetic modulator may also be cholesterol.

  Targeting moieties (conjugate moieties) are galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, N-iso-butanoylgalactosamine, galactose cluster, and N-acetyl A hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16 to 20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E), which may be selected from the group consisting of galactosamine trimers It may have a pharmacokinetic modulator selected from the group consisting of -octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clusters disclosed in '046 include (E) -hexadec-8-enoyl (C16), oleyl (C18), (9, E, 12E) -octadeca-9,12-dienoyl (C18) ), Octanoyl (C8), dodecanoyl (C12), C-20 acyl, C24 acyl, dioctanoyl (2 × C8). Targeting moiety-pharmacokinetic modulator The targeting moiety can be linked to the polynucleotide via a physiologically labile bond or, for example, a disulfide bond, or a PEG linker. The present invention also relates to the use of phosphodiester linkers between the oligomer and the conjugate group (these are referred to herein as region B and optionally located between the LNA oligomer and the carbohydrate conjugate group). .

  A preferred targeting ligand is a galactose cluster for targeting liver hepatocytes.

  Galactose clusters include molecules that have, for example, contain 2-4 terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose that have an affinity for asialoglycoprotein receptors equal to or greater than galactose. The terminal galactose derivative attaches to the molecule through its C-I carbon. The asialoglycoprotein receptor (ASGPr) is unique to hepatocytes and binds to glycoproteins with galactose at the branched ends. A preferred galactose cluster has three galactosamine or galactosamine derivatives each having affinity for the asialoglycoprotein receptor at the end. A more preferred galactose cluster is terminated with 3 N-acetylgalactosamines. Other terms common in the art include tri-branched galactose, trivalent galactose, and galactose trimer. Tri-branched galactose derivative clusters are known to bind to ASGPr with greater affinity than bi-branched or mono-branched galactose derivative structures (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al , 1982, 1. Biol. Chern., 257, 939-945). Multivalent is required to achieve nM level affinity. According to WO2012 / 083046, attachment of a single galactose derivative with affinity for the asialoglycoprotein receptor does not allow functional delivery of RNAi polynucleotides to hepatocytes in vivo when co-administered with a delivery polymer .

  A galactose cluster may comprise two or preferably three galactose derivatives each linked to a central branch point. Galactose derivatives attach to the central branch point through the C-I carbon of the saccharide. The galactose derivative is preferably linked to the branch point via a linker or spacer (which may be region Y). A preferred spacer is a flexible hydrophilic spacer (US Pat. No. 5,885,968; Biessen et al. J. Med. Chern. 1995 Vol. 39 p.1538-1546). A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer. The branch point can be any small molecule that allows attachment of the three galactose derivatives and also allows attachment of the branch point to the oligomer. An exemplary branch point group is dilysine. The dilysine molecule contains three amine groups to which three galactose derivatives can be attached and a carboxyl reactive group to which dilysine can be attached to the oligomer. Attachment of branch points to the oligomer can occur through a linker or spacer. A preferred spacer is a flexible hydrophilic spacer. A preferred flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3 spacer (3 ethylene units). Galactose clusters can be attached to the 3 ′ or 5 ′ end of the oligomer using methods known in the art.

A preferred galactose derivative is N-acetyl-galactosamine (GalNAc). Other saccharides with affinity for the asialoglycoprotein receptor can be selected from the list including galactosamine, Nn-butanoyl galactosamine, and N-iso-butanoyl galactosamine. The affinity of many galactose derivatives for the asialoglycoprotein receptor has been studied (see, eg, Jobst, STand Drickamer, K.JB.C.1996,271,6686) or methods typical in the art Is easily determined.

領域Aは、例えば、LNAアンチセンスオリゴヌクレオチドであり得る。 The GalNac conjugate is illustrated in FIG. Further examples of conjugates of the invention are illustrated below.

Region A can be, for example, an LNA antisense oligonucleotide.

  Thus, as described herein, a carbohydrate conjugate (eg, GalNAc) is optionally attached to the dilysine in the above figure via a biocleavable linker, eg, region B as defined herein. Can be linked to the oligomer via the region Y illustrated as.

  Here, the hydrophobic or lipophilic moiety (or additional conjugate moiety) (ie, pharmacokinetic modulator) in the GalNac cluster conjugate is a BNA oligomer or LNA oligomer, such as an LNA antisense oligonucleotide. Is optional.

  Specific GalNac clusters used in this study, conjugates 1, 2, 3, 4 and conjugates 1a, 2a, 3a, and 4a (shown with an optional C6 linker joining the GalNac cluster to the oligomer For-see Figures 12 and 17, see the drawings.

  Thus, each carbohydrate moiety (eg, GalNAc) of a GalNac cluster is a spacer, eg, a (poly) ethylene glycol linker (PEG), eg, a diethylene glycol linker, a triethylene glycol linker, a tetraethylene glycol linker, a pentaethylene glycol linker, a hexaethylene glycol, It can be conjugated to the oligomer via an ethylene glycol linker. As indicated above, the PEG moiety forms a spacer between the galactose sugar moiety and the peptide (trilysine shown) linker.

R¹は、-C₂H₄-、-C₃H₆-、-C₄H₈-、-C₅H₁₀-、-C₆H₁₂-、1,4-シクロヘキシル（-C₆H₁₀-）、1,4-フェニル（-C₆H₄-）、-C₂H₄OC₂H₄-、-C₂H₄(OC₂H₄)₂-、または-C₂H₄(OC₂H₄)₃-より好ましくは選択されるビラジカルである。 In some embodiments, the GalNac cluster comprises a peptide linker attached to the oligomer (or region Y or region B) via a biradical linker, such as a Tyr-Asp (Asp) tripeptide or an Asp (Asp) dipeptide, for example The GalNac cluster can include the following biradical linkers:

R ¹ _{is, -C 2 H 4 -, -} C 3 H 6 -, - C 4 H 8 -, - C 5 H 10 -, - C 6 H 12 -, 1,4- cyclohexyl (-C ₆ H ₁₀ -), 1,4-phenyl _{(-C 6 H 4 -),} - C 2 H 4 OC 2 H 4 -, - C 2 H 4 (OC 2 H 4) 2 -, or -C ₂ H ₄ (OC ₂ H ₄ ) ₃ -More preferably a biradical selected.

  In addition, the carbohydrate conjugate (eg, GalNAc) or carbohydrate-linker moiety (eg, carbohydrate-PEG moiety) can be a branch point group, eg, an amino acid, or two or more (eg, 3, 4, or 5) as appropriate. Can be covalently joined (linked) to the oligomer (or region B) via a peptide containing the amino group of, for example, lysine, dilysine or trilysine or tetralysine. The trilysine molecule is composed of three carbohydrate conjugate groups, eg galactose & derivatives (eg GalNAc) and further conjugate moieties / groups, eg 4 amines to which hydrophobic or lipophilic moieties / groups can be attached. It contains a group as well as a carboxyl reactive group to which trilysine can be attached to the oligomer. Additional conjugate moieties, such as lipophilic / hydrophobic moieties, can be attached to lysine residues attached to the oligomer. In some embodiments, the conjugate (C) is not monovalent GalNac.

  The invention also provides an LNA antisense oligonucleotide conjugated to an asialoglycoprotein receptor targeting moiety. In some embodiments, the conjugate moiety (eg, third region or region C) is an asialoglycoprotein receptor targeting moiety, eg, galactose, galactosamine, N-formyl-galactosamine, N acetylgalactosamine, N-propionyl-galactosamine Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. In some embodiments, the conjugate comprises a galactose cluster, eg, N-acetylgalactosamine trimer. In some embodiments, the conjugate moiety comprises GalNac (N-acetylgalactosamine), eg, monovalent, divalent, trivalent, or tetravalent GalNac. Trivalent GalNac conjugates can be used to target compounds to the liver. GalNac conjugates include methylphosphonate and PNA antisense oligonucleotides (eg, US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov-Dec; 6 (6): 695-701) and siRNA (eg, WO2009 / 126933, WO2012 / 089352 & WO2012 / 083046). References to GalNac and the specific conjugates used therein are incorporated herein by reference. WO2012 / 083046 discloses GalNac conjugate moieties comprising cleavable pharmacokinetic modulators, preferred pharmacokinetic modulators are C16 hydrophobic groups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E) -Octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The '046 cleavable pharmacokinetic modulator may also be cholesterol. The targeting moiety of '046 is galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, N-iso-butanoylgalactosamine, galactose cluster, and N-acetylgalactosamine A hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16 to 20 carbon atoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E)- It may have a pharmacokinetic modulator selected from the group consisting of octadeca-9,12dienoyl, dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clusters disclosed in '046 include (E) -hexadec-8-enoyl (C16), oleyl (C18), (9, E, 12E) -octadeca-9,12-dienoyl (C18) ), Octanoyl (C8), dodecanoyl (C12), C-20 acyl, C24 acyl, dioctanoyl (2 × C8). According to '046, the targeting moiety-pharmacokinetic modulator targeting moiety can be linked to the polynucleotide via a physiologically labile bond, or, for example, a disulfide bond, or a PEG linker.

  Other conjugate moieties include, for example, oligosaccharides and carbohydrate clusters, such as Tyr-Glu-Glu- (aminohexyl GalNAc) 3 (YEE (ahGalNAc) 3; glycotrins that bind to Gal / GalNAc receptors on hepatocytes. Peptides, eg, Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (eg, L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); Galactose clusters (eg, carbohydrate recognition motifs of asialoglycoprotein receptors) can be included. Further suitable conjugates can include oligosaccharides that can bind to a carbohydrate recognition domain (CRD) found on the asialoglycoprotein receptor (ASGP-R). Examples of conjugate moieties containing oligosaccharides and / or carbohydrate conjugates are provided in US Pat. No. 6,525,031, which is hereby incorporated by reference in its entirety.

Pharmacokinetic Modulators The compounds of the present invention can further comprise one or more additional conjugate moieties, for example when the conjugate group is a carbohydrate moiety, the lipophilic or hydrophobic moiety is Of particular interest. Such lipophilic or hydrophobic moieties can act as pharmacokinetic modulators, optionally linkers, eg, linkers that link carbohydrate conjugates, carbohydrate conjugates to oligomers via biocleavable linkers. Or covalently linked to a linker that links multiple carbohydrate conjugates (multivalent conjugates) to the oligomer.

  Thus, the oligomer or conjugate moiety can comprise a pharmacokinetic modulator, such as a lipophilic or hydrophobic moiety. Such moieties are disclosed for siRNA conjugates in WO2012 / 082046. The hydrophobic moiety can include C8-C36 fatty acids that can be saturated or unsaturated. In some embodiments, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, C30, C32, and C34 fatty acids may be used. Hydrophobic groups can have 16 or more carbon atoms. Exemplary suitable hydrophobic groups are selected from the group consisting of sterols, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E, 12E) -octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. obtain. According to WO'346, hydrophobic groups with less than 16 carbon atoms are less effective in enhancing polynucleotide targeting, but multiple copies (eg 2 ×, eg 2 × C8, C10, C12 , C14) can be used to enhance efficacy. Pharmacokinetic modulators useful as polynucleotide targeting moieties can be selected from the group consisting of cholesterol, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which is linear Can be shaped, branched, or annular. The pharmacokinetic modulator is preferably a hydrocarbon containing only carbon and hydrogen atoms. However, substituted or heteroatoms that maintain hydrophobicity, such as fluorine, can be tolerated.

  Surprisingly, the inventors have found that GalNac conjugates for use with LNA oligomers do not require pharmacokinetic modulators, and thus, in some embodiments, GalNac conjugates are lipophilic or It does not include hydrophobic moieties such as those described herein, such as C8-C36 fatty acids or sterols that are not covalently linked. Thus, the present invention also provides LNA oligomeric GalNac conjugates that are free of lipophilic or hydrophobic pharmacokinetic modulators or conjugate moieties / groups.

Lipophilic conjugates The compounds of the present invention may be conjugates comprising oligomer (A) and lipophilic conjugate (C). Biocleavable linkers (B) have been found to be particularly effective in maintaining or enhancing the activity of such oligomeric conjugates. In some embodiments, the conjugate group (C) and / or linker group (Y) comprises a lipophilic group.

  Exemplary conjugate moieties can include lipophilic molecules (aromatic and non-aromatic) including sterol molecules and steroid molecules. Lipophilic conjugate moieties can be used, for example, to counteract the hydrophilicity of oligomeric compounds and enhance cell entry. Lipophilic moieties include, for example, steroids and related compounds such as cholesterol (US Pat. No. 4,958,013 and Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,6553), thiocholesterol (Oberhauser et al. al, Nucl Acids Res., 1992, 20, 533), lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estratriol, progesterone, stilbestrol, testosterone, andro Examples include sterone, deoxycorticosterone, cortisone, 17-hydroxycorticosterone, and derivatives thereof.

  Other lipophilic conjugate moieties include aliphatic groups such as linear, branched, and cyclic alkyl, alkenyl, and alkynyl. An aliphatic group can have, for example, 5 to about 50, 6 to about 50, 8 to about 50, or 10 to about 50 carbon atoms. Examples of the aliphatic group include undecyl, dodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, terpene, bornyl, adamantyl, and derivatives thereof. In some embodiments, one or more carbon atoms in the aliphatic group may be substituted with a heteroatom, such as O, S, or N (eg, geranyloxyhexyl). Further suitable lipophilic conjugate moieties include aliphatic derivatives of glycerol, such as alkyl glycerol, bis (alkyl) glycerol, tris (alkyl) glycerol, monoglycerides, diglycerides, and triglycerides. In some embodiments, the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-O-hexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651). Shea, et al., Nuc. Acids Res., 1990, 18, 3777), or phosphonates thereof. Saturated and unsaturated aliphatic functionalities such as fatty acids, fatty alcohols, fatty acid esters, and aliphatic amines can also serve as lipophilic conjugate moieties. In some embodiments, the aliphatic functionality may contain about 6 to about 30 or about 8 to about 22 carbons. Examples of fatty acids include capric acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosanoic acid and the like.

  In further embodiments, the lipophilic conjugate group can be a polycyclic aromatic group having 6 to about 50, 10 to about 50, or 14 to about 40 carbon atoms. Examples of the polycyclic aromatic group include pyrene, purine, acridine, xanthene, fluorene, phenanthrene, anthracene, quinoline, isoquinoline, naphthalene, derivatives thereof and the like. Other suitable lipophilic conjugate moieties include menthol, trityl (eg, dimethoxytrityl (DMT)), phenoxazine, lipoic acid, phospholipid, ether, thioether (eg, hexyl-S-tritylthiol), their Derivatives and the like are included. The preparation of lipophilic conjugates of oligomeric compounds is described in the art, for example, Saison-Behmoaras et al, EMBO J., 1991, 10, 1111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al, Biochimie, 1993, 75, 49; Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229, and Manoharan et al., Tetrahedron Lett., 1995, 36, 3651.

  Oligomeric compounds containing conjugate moieties with affinity for low density lipoprotein (LDL) can help provide an effective target-specific delivery system. Due to the high expression level of LDL receptors on tumor cells, LDL is an attractive carrier for the selective delivery of drugs to these cells (Rump, et al., Bioconjugate Chem., 1998, 9,341; Firestone, Bioconjugate Chem., 1994, 5, 105; Mishra, et al., Biochim. Biophys. Acta, 1995, 1264, 229). Moieties that have an affinity for LDL include many lipophilic groups such as steroids (eg cholesterol), fatty acids, their derivatives, and combinations thereof. In some embodiments, the conjugate moiety having LDL affinity can be a cholic acid, eg, a dioleyl ester of chenodeoxycholic acid and lithocholic acid.

  In some embodiments, the conjugated group can be or include a lipophilic moiety, such as a sterol (eg, cholesterol, cholesteryl, cholestanol, stigmasterol, colanic acid, and ergosterol). In some embodiments, the conjugate can be or include cholesterol. See, for example, Soutschek et al., Nature (2004) 432,173; Krutzfeldt Nature 2005, NAR 2007.

  In some embodiments, conjugates are lipids, phospholipids, or lipophilic alcohols such as cationic lipids, neutral lipids, sphingolipids, and fatty acids such as stearic acid, oleic acid, elaidic acid, linoleic acid, Linoleic elaidic acid, linolenic acid, and myristic acid may be or may include. In some embodiments, the fatty acid includes a saturated or unsaturated C4-C30 alkyl chain. The alkyl chain may be linear or branched.

  In some embodiments, the lipophilic conjugate may be biotin or may include biotin. In some embodiments, the lipophilic conjugate may be a glyceride or glyceride ester or may comprise a glyceride or glyceride ester.

  Lipophilic conjugates such as cholesterol or those disclosed herein can be used to enhance delivery of oligonucleotides to, for example, the liver (typically hepatocytes).

  The following references refer to the use of lipophilic conjugates: Kobylanska et al., Acta Biochim Pol. (1999); 46 (3): 679-91; Felber et al ,. Biomaterials (2012) 33 ( 25): 599-65; Grijalvo et al., J Org Chem (2010) 75 (20): 6806-13; Koufaki et al., Curr Med Chem (2009) 16 (35): 4728-42; Godeau et al J. Med. Chem. (2008) 51 (15): 4374-6.

Polymer Conjugate The conjugate moiety can also include a polymer. The polymer can provide increased bulk and various functional groups to affect the permeation, cell transport, and localization of the conjugated oligomeric compound. For example, an increase in hydrodynamic radius caused by conjugation of oligomeric compounds with polymers can help prevent entry into the nucleus and promote localization to the cytoplasm. In some embodiments, the polymer does not substantially reduce cellular uptake and does not interfere with hybridization to a complementary strand or other target. In further embodiments, the conjugated polymer moiety has a molecular weight of, for example, less than about 40 kDa, less than about 30 kDa, or less than about 20 kDa. In addition, the polymer conjugate moiety can be water soluble and can optionally further comprise other conjugate moieties such as peptides, carbohydrates, drugs, reporter groups, or additional conjugate moieties.

  In some embodiments, the polymer conjugate includes polyethylene glycol (PEG) and copolymers and derivatives thereof. Conjugation to PEG has been shown to increase the nuclease stability of oligomeric compounds. The PEG conjugate moiety can be of any molecular weight, eg, about 100, about 500, about 1000, about 2000, about 5000, about 10,000, or more. In some embodiments, the PEG conjugate moiety is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least Contains 25 ethylene glycol residues. In further embodiments, the PEG conjugate moiety contains about 4 to about 10, about 4 to about 8, about 5 to about 7, or about 6 ethylene glycol residues. The PEG conjugate moiety may be modified, for example, the terminal hydroxyl may be substituted with alkoxy, carboxy, acyl, amide, or other functionality. Other conjugate moieties such as reporter groups such as biotin or fluorescein may be attached to the PEG conjugate moiety. Copolymers of PEG are also suitable as conjugate moieties. Preparation and biological activity of polyethylene glycol conjugates of oligonucleotides are described, for example, in Bonora, et al., Nucleosides Nucleotides, 1999, 18, 1723; Bonora, et al., Farmaco, 1998, 53, 634; Efimov, Bioorg. Khim 1993, 19, 800; and Jaschke, et al, Nucleic Acids Res., 1994, 22, 4810. Additional examples of PEG conjugate moieties and the preparation of corresponding conjugated oligomeric compounds are described, for example, in US Pat. Nos. 4,904,582 and 5,672,662, each incorporated herein by reference in their entirety. Oligomeric compounds conjugated to one or more PEG moieties are commercially available.

  Other polymers suitable as conjugate moieties include polyamines, polypeptides, polymethacrylates (eg, hydroxylpropyl methacrylate (HPMA)), poly (L-lactic acid), DL lactic acid-glycolic acid copolymer (PGLA), polyacrylic acid Polyethyleneimine (PEI), polyalkylacrylic acid, polyurethane, polyacrylamide, N-alkylacrylamide, polyspermine (PSP), polyether, cyclodextrin, derivatives thereof, and copolymers thereof. Many polymers, such as PEG and polyamines, have receptors present on certain cells, thereby facilitating cellular uptake. Polyamines and other amine-containing polymers can exist in protonated form at physiological pH, effectively counteract the anionic backbone of some oligomeric compounds and effectively enhance cell penetration . Some examples of polyamines include polypeptides (eg, polylysine, polyornithine, polyhistidine, polyarginine, and copolymers thereof), triethylenetetraamine, spermine, polyspermine, spermidine, syn norspermidine, C-branched Spermidine as well as derivatives thereof are included. The preparation and biological activity of polyamine conjugates is described, for example, in Guzaev, et al, Bioorg. Med. Chem. Lett., 1998, 8, 3671; Corey, et al, J Am. Chem. Soc, 1995, 117, 9373; and Prakash, et al, Bioorg. Med. Chem. Lett. 1994, 4, 1733. Examples of oligonucleotide polypeptide conjugates are provided, for example, in Wei, et al., Nucleic Acids Res., 1996, 24, 655 and Zhu, et al., Antisense Res. Dev., 1993, 3,265. Dendrimer polymers can also be used as conjugate moieties, for example, as described in US Pat. No. 5,714,166, which is hereby incorporated by reference in its entirety. As described above for polyamines and related polymers, other amine-containing moieties can also serve as suitable conjugate moieties, for example, for the formation of cationic species at physiological conditions. Examples of amine-containing moieties include 3-aminopropyl, 3- (N, N-dimethylamino) propyl, 2- (2- (N, N-dimethylamino) ethoxy) ethyl, 2- (N- (2- Aminoethyl) -N-methylaminooxy) ethyl, 2- (l-imidazolyl) ethyl and the like. The G-clamp moiety can also serve as an amine-containing conjugate moiety (Lin, et al., J. Am. Chem. Soc, 1998, 120, 8531).

  In some embodiments, the conjugate may be a polymer, eg, a polymer selected from the group consisting of polyethylene glycol (PEG), polyamidoamine (PAA), polyethylene oxide, and polyethyleneimine (PEI), or It may be included. Galactose, lactose, n-acetylgalactosamine, mannose, mannose-6-phosphate. In some embodiments, the polymer is a polycationic polymer. In some embodiments, the conjugate moiety may be a cationic polymer or may be based on (may include) a cationic polymer. Numerous studies have demonstrated that cationic polymers, such as cationic albumin, can greatly enhance delivery to specific cell types and / or tissues (eg, brain delivery. Lu, W. et. al. (2005) J of Control Release 107: 428-448). Because of the advantages of these molecules, the conjugate moiety can be a cationic polymer, such as polyethyleneimine, dendrimer, poly (alkylpyridinium) salt, or cationic albumin. In some embodiments, it is a hydrophilic polymer. In some embodiments, the polymer is poly (vinyl pyrrolidone) (PVP). In some embodiments, the polymer is a polyamine or polyamide (eg, US7,816,337 & US5525465). For polymer conjugates, see, for example, Zhao et al., Bioconjugate Chem 2005, 16, 758-766; Kim et al., J. Control Release (2006) 116; 123; Petetti et al., Ther. Deliv. (2011) 2 (7): 907-17; Yang et al., Bioconjug Chem (2009) 20 (2): 213-21; Winkler et al (2009) Eur J Med Chem 44 (2): 670-7; Zelikin et al, See Biomacromolecules (2007) 8 (9): 2950-3. See also WO12092373 which refers to a polynucleotide delivery conjugate cleavable by an enzyme.

Protein conjugates and peptide conjugates Other conjugate moieties may include proteins, subunits, or fragments thereof. Proteins include, for example, enzymes, reporter enzymes, antibodies, receptors and the like. In some embodiments, the protein conjugate moiety can be an antibody or fragment thereof (Kuijpers, et al, Bioconjugate Chem., 1993, 4, 94). Antibodies can be designed to bind to a desired target, such as an antigen associated with tumors and other diseases. In a further embodiment, the protein conjugate moiety can be a serum protein, such as HAS, or a glycoprotein, such as asialoglycoprotein (Rajur, et al., Bioconjugate Chem., 1997, 6, 935). In further embodiments, the oligomeric compound may be conjugated to RNAi related proteins, RNAi related protein complexes, subunits, and fragments thereof. For example, the oligomeric compound can be conjugated to Dicer or RISC. Intercalators and minor groove binders (MGB) may also be suitable as conjugate moieties. In some embodiments, the MGB may contain repeating DPI (l, 2-dihydro-3H-pyrrolo (2,3-e) indole-7-carboxylate) subunits or derivatives thereof (Lukhtanov, et al Bioconjugate Chem., 1996, 7, 564 and Afonina, et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 3199). Suitable intercalators include, for example, polycyclic aromatic compounds such as naphthalene, perylene, phenanthridine, benzophenanthridine, phenazine, anthraquinone, acridine, and derivatives thereof. Hybrid intercalator / ligand includes photonuclease / intercalator ligand 6-[[[9-[[6- (4-nitrobenzamido) hexyl] amino] acridin-4-yl] carbonyl] amino] hexanoyl-pentafluorophenyl Esters are included. This compound is an acridine moiety that is an intercalator and a p-nitrobenzamide group that is a photonuclease. In further embodiments, a cleaving agent can serve as the conjugate moiety. A cleaving agent may facilitate degradation of a target, eg, a target nucleic acid, by a hydrolysis or redox cleavage mechanism. Cleavage groups that may be suitable as conjugate moieties include, for example, constructs containing active site components of metal complexes, peptides, amines, enzymes, and nucleases, such as imidazole, guanidinium, carboxyl, amino groups, etc. Is included. Examples of metal complexes include, for example, Cu-terpyridyl complexes, Fe-porphyrin complexes, Ru complexes, and lanthanide complexes such as various Eu (III) complexes (Hall, et al., Chem. Biol, 1994). 1,185; Huang, et al., J. Biol. Inorg. Chem., 2000, 5, 85; and Baker, et al, Nucleic Acids Res., 1999, 27, 1547). Other metal complexes having cleavage properties include metalloporphyrins and their derivatives. Examples of peptides with targeted cleavage properties include zinc fingers (US Pat. No. 6,365,379; Lima, et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 10010). Examples of constructs containing nuclease active site components include bisimidazole and histamine.

  The conjugate moiety can also include a peptide. Suitable peptides can have 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 amino acid residues. Amino acid residues can be naturally occurring or non-naturally occurring, including both D and L isomers. In some embodiments, the peptide conjugate moiety is a pH sensitive peptide, eg, a fusogenic peptide. The fusogenic peptide can facilitate the release of an agent, eg, an oligomeric compound, into the cytoplasm. The fusogenic peptide is believed to change conformation at acidic pH and effectively destabilize the endosomal membrane, thereby enhancing cytoplasmic delivery of endosomal contents. Examples of the fusogenic peptide include polymycin B, influenza HA2, GALA, KALA, EALA-derived peptide, melittin-derived peptide, a-helical peptide, or Alzheimer β-amyloid peptide. The preparation and biological activity of oligonucleotides conjugated to fusogenic peptides are described, for example, in Bongartz, et al., Nucleic Acids Res., 1994, 22,4681 and US Pat. ing. Other peptides that can serve as conjugate moieties include delivery peptides that have the ability to transport relatively large polar molecules (eg, peptides, oligonucleotides, and proteins) across cell membranes. Examples of delivery peptides include Tat peptides from HIV Tat protein and Ant peptides from Drosophila antenna protein. Conjugation with Tat and Ant oligonucleotides is described, for example, in Astriab-Fisher, et al., Biochem. Pharmacol, 2000, 60, 83. These and other delivery peptides that can be used as conjugate moieties are provided in Table I below.

  Conjugated delivery peptides can help regulate the localization of oligomeric compounds to specific regions of the cell, eg, cytoplasm, nucleus, nucleolus, and endoplasmic reticulum (ER). Nuclear localization can be achieved by conjugation of a nuclear localization signal (NLS). In contrast, cytoplasmic localization can be facilitated by conjugation of nuclear export signals (NES). Peptides suitable for localization of the conjugated oligomeric compound to the nucleus include, for example, N, N-dipalmitylglycyl-apoE peptide or N, N-dipalmitylglycyl-apoE Peptides (dpG apoE) are included (Liu, et al, Arterioscler. Thromb. Vasc. Biol, 1999, 19, 2207; Chaloin, et al., Biochem. Biophys. Res. Commun., 1998, 243, 601). Localization to the nucleus or nucleolus is achieved by peptides having arginine-rich and / or lysine-rich motifs, such as HIV-1 Tat, FXR2P, and angiogenin derived peptides (Lixin, et al, Biochem. Biophys. Res. Commun., 2001, 284, 185). Furthermore, the nuclear localization signal (NLS) peptide from SV40 antigen T (Branden, et al., Nature Biotech, 1999, 17,784) can be used to deliver conjugated oligomeric compounds to the nucleus of the cell. . Other suitable peptides with localization properties to the nucleus or nucleolus are described in, for example, Antopolsky, et al., Bioconjugate Chem., 1999, 10, 598; Zanta, et al., Proc. Natl. Acad. Sci. USA, 1999 (monkey virus 40 large tumor antigen); Hum. Mol. Genetics, 2000, 9, 1487; and FEBSLett., 2002, 532, 36.

  In some embodiments, the delivery peptide for localization to the nucleus or nucleolus comprises at least 3 consecutive arginine residues or at least 4 consecutive arginine residues. Nuclear localization can also be facilitated by peptide conjugates containing RS repeat motifs, RE repeat motifs, or RD repeat motifs (Cazalla, et al., Mol Cell. Biol, 2002, 22, 6871). . In some embodiments, the peptide conjugate contains at least two RS motifs, RE motifs, or RD motifs.

  For example, the localization of oligomeric compounds to the ER can be achieved by the signal peptide KDEL (SEQ ID NO: 18) (Arar, et al., Bioconjugate Chem., 1995, 6,573; Pichon, et al., Mol. Pharmacol. 1997, 57,431). Cytoplasmic localization of oligomeric compounds can be facilitated, for example, by conjugation to peptides with nuclear export signals (NES) (Meunier, et al., Nucleic Acids Res., 1999, 27, 2730). NES peptides include leucine-rich NES peptides derived from HIV-1 Rev (Henderson, et al., Exp. Cell Res., 2000, 256, 213), transcription factor III A, MAPKK, PKI-α, cyclin Bl, and actin ( Wada, et al., EMBO J., 1998, 17, 1635), and related proteins. Antimicrobial peptides such as dermaseptin derivatives can also facilitate cytoplasmic localization (Hariton-Gazal, et al., Biochemistry, 2002, 41, 9208). Peptides containing RG repeat motifs and / or KS repeat motifs may also be suitable for directing oligomeric compounds to the cytoplasm. In some embodiments, the peptide conjugate moiety contains at least 2 RG motifs, at least 2 KS motifs, or at least 1 RG motif and 1 KS motif. As used herein, “peptide” includes not only the specific molecules or sequences described herein (if any), but also fragments thereof that retain the desired functionality, Also included are molecules that contain all or part of the described sequences. In some embodiments, the peptide fragment contains 6 or more amino acids. A peptide may contain conservative amino acid substitutions that do not substantially change its functional characteristics. Conservative substitutions can be made in the following sets of functionally similar amino acids: neutral-weakly hydrophobic (A, G, P, S, T), hydrophilic-acidic amines (N, D, Q , E), hydrophilic-basic (I, M, L, V), and hydrophobic-aromatic (F, W, Y). Peptides include homologous peptides. Homology can be measured, for example, by percent identity using the BLAST algorithm (default parameters for short sequences). For example, homologous peptides can have greater than 50, 60, 70, 80, 90, 95, or 99 percent identity. Methods for conjugating peptides to oligomeric compounds such as oligonucleotides are described, for example, in US Pat. No. 6,559,279, which is hereby incorporated by reference in its entirety.

  In some embodiments, the conjugate moiety is or includes a protein or peptide. In some embodiments, the peptide is a cell-invading peptide, such as penetratin, transportan, peptibol (eg, trichorovin-XIIa (TV-XIIa)), TAT peptide (HIV). In some embodiments, the peptide is a polyarginine (eg, stearyl- (RxR) (4)). In some embodiments, the peptide is N- (2-hydroxypropyl) methacrylamide (HPMA) containing the tetrapeptide Gly-Phe-Leu-Gly (GFLG). In some embodiments, the peptide is a β-amyloid peptide. In some embodiments, the protein or peptide is an antibody or antigen binding site-containing fragment thereof (epitope binding site). In some embodiments, the conjugate is or comprises M6P-HPMA-GFLG (see Yang et al 2009). In some embodiments, the conjugate is or comprises an arginine rich peptide (WO2005 / 115479). See also WO09005793 (RGD peptide). In some embodiments, the conjugate is or includes a protein carrier (eg, albumin, albumin-PEG conjugate, RGD-PEG-albumin) (Kang et al). See also WO09045536. In some embodiments, the conjugate is or comprises a histidylated oligolysine (eg, WO0032764). In some embodiments, the conjugate is or includes a glycoprotein: transferrin-polycation (eg, US5354844, WO9217210, WO9213570). In some embodiments, the conjugate is or comprises an asialoglycoprotein (US5346696). In some embodiments, the conjugate is or comprises a polycationic protein (eg, US603095). In some embodiments, the conjugate is or comprises a poly-pseudo-lysine conjugate (eg, WO07113531).

Reporter groups and dye conjugate groups Reporter groups that are suitable as conjugate moieties include, for example, any moiety that can be detected by spectroscopic means. Examples of reporter groups include dyes, fluorophores, phosphors, radiolabels and the like. In some embodiments, the reporter group is biotin, fluorescein, rhodamine, coumarin, or related compounds. The reporter group may be attached to other conjugate moieties. In some embodiments, the conjugate is or includes a label or dye, eg, a fluorophore, eg, FAM (carboxyfluorescein).

  Crosslinkers can also serve as conjugate moieties. Crosslinkers facilitate covalent linkage of conjugated oligomeric compounds with other compounds. In some embodiments, the cross-linking agent can covalently link double-stranded nucleic acids, effectively increasing duplex stability and modulating pharmacokinetic properties. In some embodiments, the cross-linking agent can have photoactivity or redox activity. Examples of cross-linking agents include psoralen (Lin, et al, Faseb J, 1995, 9, 1371) that can facilitate interstrand cross-linking of nucleic acids by photoactivation. Other cross-linking agents include, for example, mitomycin C and analogs thereof (Maruenda, et al., Bioconjugate Chem., 1996, 7,541; Maruenda, et al., Anti-Cancer Drug Des., 1997, 12,473; and Huh , et al, Bioconjugate Chem., 1996, 7,659). Mitomycin C mediated cross-linking can be achieved by reductive activation, eg, biological reducing agents (eg, NADPH-cytochrome c reductase / NADPH system). Additional photocrosslinkers include, for example, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and N-succinimidyl-6 (-4′-azido-2′-nitrophenyl-amino) hexanoate (SANPAH). Aryl azides are included. Aryl azides conjugated to oligonucleotides achieve cross-linking with nucleic acids and proteins upon irradiation. They also crosslink with an earner protein (eg KLH or BSA).

Various functional conjugate groups Other suitable conjugate moieties include, for example, polyborane, carborane, metallopolyborane, metallocarborane, derivatives thereof, etc. (eg, incorporated herein by reference in its entirety). U.S. Pat. No. 5,272,250).

  Many drugs, receptor ligands, toxins, reporter molecules, and other small molecules can serve as conjugate moieties. Small molecule conjugate moieties often interact specifically with certain receptors or other biomolecules, thereby enabling the targeting of conjugated oligomeric compounds to specific cells or tissues To. Examples of small molecule conjugate moieties include mycophenolic acid (inosine-5'-monophosphate dihydrogenase inhibitor; useful for treating psoriasis and other skin disorders), curcumin (psoriasis, cancer, bacterial With therapeutic application to diseases, and viral diseases). In a further embodiment, the small molecule conjugate moiety may be a serum protein, eg, a ligand for human serum albumin (HSA). A number of ligands for HSA are known, such as arylpropionic acid, ibuprofen, warfarin, phenylbutazone, suprofen, capprofen, fenfufen, ketoprofen, aspirin, indomethacin, (S)-(+)-planopro Phen, dansyl sarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepine, indomethicin, barbituric acid, cephalosporins, sulfa drugs, Including antibacterial drugs, antibiotics (eg, puromycin and paramycin) and the like. Oligonucleotide-drug conjugates and their preparation are described, for example, in WO00 / 76554, which is incorporated herein by reference in its entirety.

  In some embodiments, the conjugate is or can include a small molecule, eg, a small molecule drug or small molecule prodrug. Certain drugs are highly effective for targeting specific target tissues or cells, so they can be used to target oligonucleotides to their intended site of action. . In addition, small molecules typically may have therapeutic activity themselves after being cleaved from the oligonucleotide component of the conjugate. Examples include bisphosphonates (which are widely used for the treatment of osteoporosis and are effective for targeting bone tissue), anticancer drugs, and chemotherapeutic agents (eg, doxorubicin or mitomycin C-US5776907 ) Is included. In some embodiments, the drug can be a nucleoside analog, eg, a nucleoside polymerase inhibitor.

  In further embodiments, the small molecule conjugate can target or bind to a particular receptor or cell. T cells are known to have exposed amino groups that can form Schiff base complexes with appropriate molecules. Thus, functional groups that can interact with or react with exposed amino groups, such as small molecules containing aldehydes, may also be suitable conjugate moieties. Tucaresol and related compounds can be conjugated to the oligomeric compound in such a way that the aldehyde is free to interact with the T cell target. It is believed that the interaction of tukaresole with T cells results in therapeutic enhancement of the immune system by Schiff base formation (Rhodes, et al., Nature, 1995, 377, 6544).

  In some embodiments, the conjugate is or includes a receptor ligand (eg, a cell surface). In some embodiments, the conjugate is or comprises a folate receptor ligand, such as a folate group (see, eg, EP1572067 or WO2005 / 069994, WO2010 / 045584). Other cell surface receptor ligands include antibodies and fragments thereof, prostate specific membrane antigens, neuronal surface antigens (see WO2011 / 131693).

  In some embodiments, the conjugate moiety can be a ligand for a receptor or (next) bound to a molecule that binds to the receptor. This class includes bile acids, small molecule drug ligands, vitamins, aptamers, carbohydrates, peptides (including but not limited to hormones, proteins, protein fragments, antibodies, or antibody fragments), viral proteins (eg, capsids) , Toxins (eg, bacterial toxins) and the like. This class also includes steroidal conjugates such as cholesterol, cholestanol, colanic acid, stigmasterol, pregnolones, progesterone, corticosterone, aldosterone, testosterone, estradiol, ergosterol and the like. Preferred conjugate moieties of the present disclosure are cholesterol (CHOL), cholestanol (CHLN), colanic acid (CHLA), stigmasterol (STIG), and ergosterol (ERGO). In certain preferred embodiments, the conjugate moiety is cholesterol.

In some embodiments, the conjugate includes a sterol, such as cholesterol or tocopherol, and optionally includes a linker, such as a fatty acid linker, such as a C6 linker. In some embodiments, the conjugate includes conjugate 5a or conjugate 6a.

  The conjugate moiety can also include vitamins. Vitamins are known to be transported to cells by a number of cellular transport systems. Typically, vitamins can be classified as water soluble or fat soluble. Water-soluble vitamins include thiamine, riboflavin, nicotinic acid or niacin, vitamin B6 pyridoxal group, pantothenic acid, biotin, folic acid, B] 2 cobamide coenzyme, inositol, choline, and ascorbic acid. Fat-soluble vitamins include the vitamin A family, vitamin D, vitamin E tocopherol family, and vitamin K (and phytol). Related compounds include retinoid derivatives such as tazarotene and etretinate. In some embodiments, the conjugate moiety includes folic acid (folate) and / or one or more of various forms thereof, such as dihydrofolic acid, tetrahydrofolic acid, folinic acid, ptero polyglutamic acid, dihydro Folate, tetrahydrofolate, tetrahydropterin, 1-deaza analogue, 3-deaza analogue, 5-deaza analogue, 8-deaza analogue, 10-deaza analogue, 1,5-dideaza analogue of folate 5,10-dideaza analogs, 8,10-dideaza analogs, and 5,8-dideaza analogs, and antifolates. Folate is involved in nucleic acid biosynthesis and thus affects cell survival and proliferation. Folate cofactor plays a role in the one-carbon transfer required for the biosynthesis of pyrimidine nucleosides. Thus, cells have a system for transporting folate to the cytoplasm. Folate receptors also tend to be overexpressed in many human cancer cells, and folate-mediated targeting of oligonucleotides to ovarian cancer cells has been reported (incorporated herein by reference in its entirety). Li, et al, Pharm. Res. 1998, 15, 1540). The preparation of nucleic acid folate conjugates is described, for example, in US Pat. No. 6,528,631, which is incorporated herein by reference in its entirety.

  Vitamin conjugate moieties include, for example, vitamin A (retinol) and / or related compounds. Vitamin A family (retinoids), including retinoic acid and retinol, are typically specific proteins, such as cytosolic retinol binding protein type II (CRBP-II), retinol binding protein (RBP), and cellular retinol binding Through interaction with the protein (CRBP), it is absorbed and transported to the target tissue. The vitamin A family of compounds can be attached to oligomeric compounds via the acid or alcohol functionality found in various family members. For example, conjugation of the N-hydroxysuccinimide ester of the acid moiety of retinoic acid to the amine function on the linker attached to the oligonucleotide will link the vitamin A compound to the oligomeric compound via an amide bond. Can bring. Retinol may be converted to its phosphoramidite that is useful for 5 ′ conjugation. α-Tocopherol (vitamin E) and other tocopherols (β-ζ) can be conjugated to oligomeric compounds to enhance uptake by lipophilic characteristics. Vitamin D and its ergosterol precursor can also be conjugated to the oligomeric compound through the hydroxyl group by first activating the hydroxyl group, eg, to a hemisuccinate. Conjugation can then be achieved directly with the oligomeric compound, or an amino linker attached to the oligomeric compound. Other vitamins that can be similarly conjugated to the oligomeric compounds include thiamine, riboflavin, pyridoxine, pyridoxamine, pyridoxal, deoxypyridoxine. Fat-soluble vitamin K and related quinone-containing compounds can be conjugated via a carbonyl group on the quinone ring. The phytol portion of vitamin K can also serve to enhance the binding of oligomeric compounds to cells.

  Other functional groups that can be used as conjugates in the compounds of the present invention include imidazole conjugates-RNase A catalytic center mimics (polyamine-imidazole conjugates) (Guerniou et al Nucleic Acids Res (2007); 35 (20): 6778-87).

  The conjugate is typically a non-nucleotide moiety. However, with respect to blocking or targeting groups, or nucleotide analog small molecule therapeutics, it will be appreciated that the oligonucleotide can be covalently linked to the nucleotide moiety via the DNA / RNA phosphodiester region of the present invention. Optionally, the nucleic acid group used in connection with the present invention may lack complementarity to the target of the oligonucleotide (region A) in some embodiments.

  In some embodiments, the blocking or targeting moiety is an aptamer (see, eg, Meng et al., PloS (2012) 7 (4): e33434, WO2005 / 111238 & WO12078637).

  The blocking group may be or may include an antisense oligonucleotide, eg, an oligonucleotide region that is complementary to a portion of the antisense oligonucleotide. In this regard, the blocking oligonucleotide is covalently attached to the antisense oligonucleotide via the DNA / RNA phosphodiester region (region b), optionally via a linker. Thus, blocking oligonucleotides can form duplexes with antisense oligonucleotides in some embodiments. Optionally, the blocking nucleotide sequence (as the third region or region C) forms a duplex with (ie is complementary to) the first region of equal length, eg a short oligo of 3-10 nucleotides in length. Nucleotide sequence. In some embodiments, a linker is used between the second region and the blocking region.

  Similar to delivery peptides, nucleic acids can serve as conjugate-like moieties that can affect the localization of the conjugated oligomeric compound in the cell. For example, the nucleic acid conjugate moiety can contain poly A, a motif recognized by poly A binding protein (PABP) that localizes poly A containing molecules to the cytoplasm (Gorlach, et al., Exp. Cell Res., 1994, 211, 400). In some embodiments, the nucleic acid conjugate moiety is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 Contains at least 25 consecutive A bases. The nucleic acid conjugate portion may contain one or more AU rich sequence elements (ARE). ARE is recognized by ELAV family proteins that can facilitate localization to the cytoplasm (Bollig, et al, Biochem. Bioophys. Res. Commun., 2003, 301, 665). Examples of AREs include UUAUUUAUU and sequences containing multiple repeats of this motif. In other embodiments, the nucleic acid conjugate moiety contains two or more AU motifs or AUU motifs. Similarly, a nucleic acid conjugate moiety is a CU rich sequence element (CRE) that can bind to the proteins HuD and / or HuR of the ELAV family of proteins (Wein, et al, Eur. J. Biochem., 2003, 270, 350). One or more of them may be contained. Similar to ARE, CRE can help localize conjugated oligomeric compounds to the cytoplasm. In some embodiments, the nucleic acid conjugate moiety contains a motif (CUUU) n (eg, n can be 1 to about 20, 1 to about 15, or 1 to about 11). Optionally, one or more U may be present before or after the (CUUU) n motif. In some embodiments, n is about 9 to about 12 or about 11. Nucleic acid conjugate moieties can also include substrates for hnRNP proteins (heteronuclear ribonucleoproteins), some of which are involved in nucleic acid round trips between the nucleus and the cytoplasm (eg, nhRNP Al, nhRNP K; see, for example, Mili, et al, Mol. Cell Biol, 2001, 21, 7307). Some examples of hnRNP substrates include nucleic acids containing the sequence UAGGA / U or (GG) ACUAGC (A). Other nucleic acid conjugate moieties can include a Y string or other tract that can bind to, for example, linRNP I. In some embodiments, the nucleic acid conjugate is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 , And at least 25 consecutive pyrimidine bases. In other embodiments, the nucleic acid conjugate may contain greater than 50 percent, greater than 60 percent, greater than 70 percent, greater than 80 percent, greater than 90 percent, or greater than 95 percent pyrimidine base.

  Other nucleic acid conjugate-like moieties may include a pumilio (puf protein) recognition sequence as described, for example, in Wang, et al., Cell, 2002, 110,501. An example of a pumilio recognition sequence can include UGUANAUR, where N can be any base and R can be a purine base. Cytoplasmic localization can be facilitated by nucleic acid conjugate moieties containing ARE and / or CRE. Nucleic acid conjugate-like moieties that serve as substrates for hnRNP can facilitate localization of the conjugated oligomeric compound to the cytoplasm (eg, hnRNP Al or K) or nucleus (eg, hnRNP I). Furthermore, nuclear localization can be facilitated by nucleic acid conjugate-like moieties that contain polypyrimidine tracts.

Reactive Group In the context of the present invention, a reactive group is intended to “conjugate” an oligonucleotide to a third region (X), eg, a conjugate group, blocking group, or targeting group, or optionally a linker (Y), or A group used in chemical synthesis that can be used to covalently link nucleotides. An example of a reactive group is a phosphoramidite that is widely used in oligonucleotide synthesis.

Activating Group An activating group is a group that can be activated to form a reactive group. In this regard, an activating group is considered a protected reactive group that can be pre-deprotected to allow the use of the reactive group, for example, in the synthetic / manufacturing methods disclosed herein. obtain.

Linking Group A nucleoside linkage can be a linking group between nucleosides within an oligonucleotide, or it can mean a group that, when present, attaches a third region (X or C) or linker (Y) to region B. For example, the linker can be a phosphate (containing) linking group or a triazole group.

Blocker group (also called blocking / blocker part)
In some aspects, the third region is a blocking region. The blocker typically prevents or reduces the activity of the oligomer, for example through (but not limited to) steric hindrance or through hybridization with the first region (or the first and second regions). The conjugate or oligonucleotide to be made (typically not complementary to the target region). (Blocked) activity may be against its intended target, or in some embodiments, against an unintended target (off-target) Also good.

  Thus, an oligomeric compound of the invention comprises a first region, eg, a gapmer or LNA gapmer oligonucleotide (eg, a gapmer of formula X′Y′Z) and a second region that is a biocleavable linker, eg, Region B described herein and at least two consecutive nucleosides that are complementary to the corresponding components of the first region, e.g., 3, 4, 5, 6, 7, 8, A region C that is a third region comprising a region of 9, 10, 11, 12, 13, 14, 15, 15 nucleotides. In some embodiments, at least two nucleosides of region C, such as 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, are high affinity nucleoside analogues. Body, eg, LNA (BNA), and in some embodiments they may form the distal component of region C. The high affinity nucleoside analog of region C may form a continuous sequence of high affinity nucleoside analogs, to which other nucleosides such as DNA nucleosides (also part of region C, region Called the proximal component of C) may be adjacent. In some embodiments, region C comprises 2-8, such as 3, 4, 5, 6, 7 LNA (BNA) nucleotides, and in the same or different embodiments, 2-16 Contains a region of DNA nucleotides (eg 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . In some embodiments, the distal component of region B is a continuous region of high affinity nucleotide analogs, such as 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides. Of continuous regions. The proximal region may include non-LNA nucleotides, such as those referred to herein, for example, a continuous region of DNA nucleotides, such as a region of 2-16 non-LNA nucleotides. However, it is also understood that the proximal region can contain high affinity nucleotide analogs including LNA, but the contiguous region of LNA can limit the structural flexibility of the proximal region (which is thought to act as a loop). Is done. In some embodiments, it may be useful to limit the use of long stretches of LNA within the proximal (or loop-forming component) (eg, no more than 4 consecutive LNAs, eg, no more than 3 consecutive LNAs, Or two or less continuous LNA).

  In some embodiments, regions of other nucleotides (eg, DNA nucleotides) in region C form a contiguous sequence with region B, ie, are proximal to the terminal nucleotides of region B, so high affinity The region of nucleotides is distal to region B. In such an embodiment, the proximal component of region B and region C (eg, the region comprising DNA nucleotides) can form a flexible loop, which causes the distal component of region C to interact with the first region. Allows to hybridize. The proximal component of region C may or may not be complementary to the corresponding component of region A. In some embodiments, the distal component of region C is complementary to a nucleotide that forms a region capable of recruiting RNaseH, eg, a gap region of a gapmer (referred to herein as region Y ′). . In such embodiments, the blocking region (region C) forms a duplex with the gap region or part thereof, thereby central region of the gapmer for interacting with other molecules or targets or off-targets Prevent the availability of. Thus, the present invention is typically used for DNA phosphorothioate oligonucleotides (gapmer gap regions) to allow for regulated activation of gapmer oligomers (region A) in target tissues or cells. ) To solve the inherent toxicity. In this regard, the use of the blocking region can act as a prodrug. The blocking region (region C or its distal component) is for other regions of the oligomer, including mixmer or totalmer oligomers, or adjacent regions of the gapmer, or regions that span the gapmer's wing and gap regions. It is recognized that it may be. In such embodiments, hybridization of region C (or its distal component) to region A (or a portion of region A) prevents hybridization of the corresponding component of region A to the biomolecule, and thus It can also be used to prevent unintentional interactions with other biomolecules, enhance specificity, tissue-specific activity, and reduce the risk of toxicity. The internucleoside linkage between the nucleotides in region C can be other than a phosphodiester, for example a phosphorothioate.

Targeting group (also called targeting moiety)
A targeting moiety is a group that, when present on an oligomeric compound, causes a differential pattern of biodistribution and / or cellular uptake of the oligomeric compound. The targeting group can be, for example, a receptor ligand, antibody, hormone, or hormone analog, aptamer, and the like. The examples show the use of cholesterol as a targeting group. Cholesterol is recognized by the LDL receptor on the surface of hepatocytes, resulting in preferential uptake of the cholesterol-conjugated oligonucleotide into the liver. The examples also illustrate the use of GalNac, tocopherol, and folic acid as targeting groups.

Biocleavable conjugate linked to an oligomer The oligomeric compound may optionally comprise a second region (region B) located between the oligomer (referred to as region A) and the conjugate (referred to as region C). Region B can be a linker, such as a cleavable linker (also referred to as a physiologically labile linkage).

  In some embodiments, the compounds of the invention comprise a biocleavable linker (a physiologically labile linker, nuclease sensitive) that joins an oligomer (or continuous nucleotide sequence or region A) to a conjugate moiety (or region C). Physiologically labile linkages, also referred to as nuclease-sensitive linkers), eg, phosphate nucleotide linkers (eg, region B) or peptide linkers.

  A biocleavable linker according to the present invention refers to a linker that is sensitive to cleavage (ie, physiologically unstable) in the target tissue, eg, liver and / or kidney. The cleavage rate found in the target tissue is preferably greater than that found in serum. A suitable method for determining the level of cleavage (%) in tissue (eg, liver or kidney) and serum is found in Example 6. In some embodiments, a biocleavable linker (also referred to as a physiologically labile linker or nuclease sensitive linker) in the compounds of the invention, eg, region B, is at least in the liver or kidney homogenate assay of Example 6. About 20% cut, eg, at least about 30% cut, eg, at least about 40% cut, eg, at least about 50% cut, eg, at least about 60% cut, eg, at least about 70% cut For example, it is cut at least about 75%. In some embodiments, the percent cleavage in serum used in the assay in Example 6 is less than about 20%, such as less than about 10%, such as less than 5%, such as less than about 1%.

  A biocleavable linker according to the present invention refers to a linker that is sensitive to cleavage (ie, physiologically unstable) in the target tissue, eg, liver and / or kidney. The cleavage rate found in the target tissue is preferably greater than that found in serum. A suitable method for determining the level of cleavage (%) in tissue (eg, liver or kidney) and serum is found in Example 6. In some embodiments, a biocleavable linker (also referred to as a physiologically labile linker or nuclease sensitive linker) in the compounds of the invention, eg, region B, is at least in the liver or kidney homogenate assay of Example 6. About 20% cut, eg, at least about 30% cut, eg, at least about 40% cut, eg, at least about 50% cut, eg, at least about 60% cut, eg, at least about 70% cut For example, it is cut at least about 75%. In some embodiments, the percent cleavage in serum used in the assay in Example 6 is less than about 30%, less than about 20%, such as less than about 10%, such as less than 5%, such as Less than about 1%.

  In some embodiments, which may be the same or different, a biocleavable linker (also referred to as a physiologically labile linker or nuclease sensitive linker) in the compounds of the invention, eg, region B is Sensitive to S1 nuclease cleavage. Sensitivity to S1 cleavage can be assessed using the S1 nuclease assay shown in Example 6. In some embodiments, a biocleavable linker (also referred to as a physiologically labile linker or a nuclease sensitive linker) in the compounds of the invention, eg, region B is bound to S1 nuclease according to the assay used in Example 6. After 120 minutes incubation, is at least about 30% cleaved, such as at least about 40% cleaved, such as at least about 50% cleaved, such as at least about 60% cleaved, such as at least about 70% cleaved, For example, at least about 80% cut, such as at least about 90% cut, such as at least 95% cut.

Nuclease sensitive physiologically labile linkages: In some embodiments, the oligomers (also referred to as oligomeric compounds), (or conjugates) of the invention comprise the following three regions:
(I) a first region (region A) comprising 10-18 contiguous nucleotides;
(Ii) Second region containing a biocleavable linker (region B)
(Iii) A third region (C) comprising a conjugate moiety, a targeting moiety, and an activating moiety and covalently linked to the second region.

  In some embodiments, region B can be a phosphate nucleotide linker. For example, such linkers can be used when the conjugate is a lipophilic conjugate, such as a lipid, fatty acid, sterol, such as cholesterol or tocopherol. The phosphate nucleotide linker can also be used for other conjugates, such as carbohydrate conjugates, such as GalNac.

Peptides and other linkers In some embodiments, the biocleavable linker (region B) is a peptide that can be used in a poly GalNac conjugate, such as a tri-GalNac conjugate, such as a trilysine peptide linker. Other linkers known in the art can also be used, including disulfide linkers (also referred to herein as dithio or disulfide). Other peptide linkers include, for example, Tyr-Asp (Asp) tripeptide or Asp (Asp) dipeptide.

Phosphate nucleotide linker In some embodiments, region B is 1-6 covalently linked to the 5 ′ or 3 ′ nucleotide of the first region, for example, via an internucleoside linking group, such as a phosphodiester linkage. Comprising nucleotides, where
(A) The internucleoside linkage between the first region and the second region is a phosphodiester linkage, and the nucleoside in the second region adjacent [eg directly] to the first region is either DNA or RNA. And / or (b) at least one nucleoside of the second region is a DNA or RNA nucleoside linked by a phosphodiester.

  In some embodiments, region A and region B form a single contiguous nucleotide sequence that is 10-22 nucleotides long, eg, 12-20 nucleotides long.

  In some aspects, the internucleoside linkage between the first region and the second region can be considered part of the second region.

Linker A linker or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. . The conjugate moiety (or targeting moiety or blocking moiety) can be attached to the oligomeric compound directly or through a linking moiety (linker or tether). A linker is a bifunctional moiety that serves to covalently connect a third region, eg, a conjugate moiety, to an oligomeric compound (eg, region B). In some embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol units or amino acid units. The linker can have at least two functionalities, one for attachment to the oligomeric compound and the other for attachment to the conjugate moiety. Examples of linker functionality can be electrophilic to react with nucleophilic groups on the oligomer or conjugate moiety, or nucleophilic to react with electrophilic groups. In some embodiments, linker functionality includes amino, hydroxyl, carboxylic acid, thiol, phosphoramidate, phosphate, phosphite, unsaturation (eg, double bond or triple bond), and the like. Some examples of linkers include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid ( AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4- (N-maleimidomethyl) cyclohexane-l-carboxy- (6-amido-caproate) (LCSMCC), succinimidyl m-maleimide-benzoylate (MBS), succinimidyl Ne-maleimide-caproylate (EMCS), succinimidyl 6- (β-maleimide-propionamido) hexanoate (SMPH), succinimidyl N- (a-maleimide acetate) (AMAS), succinimidyl 4 -(p-Maleimidophenyl) butyrate (SMPB), β-alanine (β-ALA), phenylglycine (PHG), 4-aminocyclohexa Acid (ACHC), beta-(cyclopropyl) alanine (β-CYPR), aminododecanoic acid (ADC), alkylene diol, polyethylene glycol, amino acids or the like.

  A variety of additional linker groups are known in the art that may be useful in attaching conjugate moieties to oligomeric compounds. Many reviews of useful linker groups can be found, for example, in Antisense Research and Applications, S. T. Crooke and B. Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. Disulfide linkage has been used to link the 3 ′ end of oligonucleotides to peptides (Corey, et al., Science 1987, 238, 1401; Zuckermann, et al, J Am. Chem. Soc. 1988, 110, 1614; and Corey, et al., J Am. Chem. Soc. 1989, 111, 8524). Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe ligation reagents for attaching biotin to the 3 ′ end of oligonucleotides. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol, is commercially available from Clontech Laboratories (Palo Alto, Calif.) Under the name 3'-Amine. It is also commercially available from Glen Research Corporation (Sterling, Va.) Under the name 3′-Amino-Modifier reagent. This reagent was also utilized to link peptides to oligonucleotides as reported by Judy, et al., Tetrahedron Letters 1991, 32,879. A similar commercially available reagent for linking to the 5 ′ end of the oligonucleotide is 5′-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or the like were utilized by Krieg, et al, Antisense Research and Development 1991, 1,161 to link fluorescein to the 5 ′ end of the oligonucleotide. Other compounds, such as acridine, were attached to the 3′-terminal phosphate group of the oligonucleotide via a polymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297). . Any of the above groups can be used as a single linker or in combination with one or more additional linkers.

  Linkers and their use in preparing conjugates of oligomeric compounds are known in the art, for example, WO96 / 11205 and WO98 / 52614 and US Pat. Nos. 4,948,882; 5,525,465, each incorporated by reference in their entirety; 5,541,313; 5,545,730; 5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469; 5,245,022; 5,112,963; No. 5,510,475; No. 5,512,667; No. 5,574,142; No. 5,684,142; No. 5,770,716; No. 6,096,875; No. 6,335,432; and No. 6,335,437.

  As used herein, a physiologically labile bond is a labile bond that is cleavable under conditions normally found in or similar to the mammalian body (also called a cleavable linker). be called). A physiologically labile linking group is selected to undergo chemical transformation (eg, cleavage) when present in certain physiological conditions. Conditions within mammalian cells are similar to or similar to chemical conditions found within mammalian cells, such as pH, temperature, oxidizing or reducing conditions, oxidizing or reducing agents, and salt concentrations. Conditions are included. Conditions within mammalian cells also include the presence of enzymatic activity normally present in mammalian cells, eg, from proteolytic enzymes or hydrolases. In some embodiments, the cleavable linker is sensitive to, for example, a nuclease that can be expressed in the target cell, and thus, as detailed herein, the linker is, for example, 1-10 It can be a short region of nucleosides linked by phosphodiester, for example DNA nucleosides.

  Chemical deformation (cleavage of the labile bond) can be initiated by the addition of a pharmaceutically acceptable agent to the cell, or the molecule containing the labile bond can be appropriately intracellular and / or cellular. It can happen naturally when it reaches the outside environment. For example, a pH labile bond can be cleaved when a molecule enters an acidified endosome. Thus, pH labile bonds can be considered as endosomal cleavable bonds. Enzymatic cleavable bonds can be cleaved when exposed to enzymes such as those present in endosomes or lysosomes or cytoplasm. Disulfide bonds can be broken as the molecule enters the more reducing environment of the cytoplasm. Thus, disulfides can be considered as cytoplasmic cleavable bonds. As used herein, a pH labile bond is a labile bond that is selectively degraded under acidic conditions (pH <7). Since cellular endosomes and lysosomes have a pH of less than 7, such binding can also be termed endosomal labile binding.

Activated oligomers In some embodiments, the present invention provides intermediates used in the synthesis of activated oligomers, ie, oligomers of the present invention, eg, conjugated oligomers. In this regard, the oligomer of the present invention may in some embodiments comprise region A and region B as described herein, wherein region B is a suitable activity for use in oligomer conjugation. It is covalently linked to a chemical (or reactive) group.

As used herein, the term “activated oligomer” refers to one or more conjugated moieties, ie nucleic acids or monomers themselves, to form the conjugates described herein. An oligomer of the present invention that is covalently linked (ie, functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to a non-part. Typically, the functional group moiety is, for example, a 3 ′ hydroxyl group or exocyclic NH ₂ group of an adenine base, preferably a spacer that is hydrophilic, and a terminal group that can be attached to the conjugated moiety (eg, , Amino groups, sulfhydryl groups, or hydroxyl groups) will contain chemical groups that can be covalently attached to the oligomer. In some embodiments, this end group is unprotected, eg, an NH ₂ group. In other embodiments, the end groups are, for example, by suitable protecting groups such as those described in “Protective Groups in Organic Synthesis” by Theodora W Greene and Peter GM Wuts, 3rd edition (John Wiley & Sons, 1999). Protected. Examples of suitable hydroxyl protecting groups include esters such as acetate esters, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, α-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoro Contains acetyl. In some embodiments, the functional group moiety is self-cleavable. In other embodiments, the functional moiety is biodegradable. See, eg, US Pat. No. 7,087,229, which is incorporated herein by reference in its entirety.

  In some embodiments, the oligomer of the invention is functionalized at the 5 ′ end to allow covalent attachment of the conjugated moiety to the 5 ′ end of the oligomer. In other embodiments, the oligomers of the invention may be functionalized at the 3 ′ end. In yet other embodiments, the oligomers of the invention are functionalized along the backbone or at the heterocyclic base moiety. In still other embodiments, the oligomers of the invention may be functionalized at a plurality of positions independently selected from the 5 ′ end, 3 ′ end, backbone, and base.

In some embodiments, activated oligomers of the invention are synthesized by incorporating one or more monomers covalently attached to the functional moiety during synthesis. In other embodiments, the activated oligomers of the present invention are synthesized with unfunctionalized monomers, and the oligomers are functionalized after completion of the synthesis. In some embodiments, the oligomer comprises an aminoalkyl linker in which the alkyl moiety has the formula (CH ₂ ) _w , where w is an integer ranging from 1 to 10, preferably about 6. Functionalized with hindered esters. The alkyl portion of the alkylamino group can be linear or branched and the functional group is attached to the oligomer via an ester group (—OC (O) — (CH ₂ ) _w NH).

In other embodiments, the oligomer comprises a hindered ester containing a (CH ₂ ) _w -sulfhydryl (SH) linker, wherein w is an integer in the range of 1-10, preferably about 6. Is functionalized by The alkyl part of the alkylamino group can be linear or branched and the functional group is attached to the oligomer via an ester group (—OC (O) — (CH ₂ ) _w SH).

  In some embodiments, the sulfhydryl activated oligonucleotide is conjugated to a polymer moiety, eg, polyethylene glycol or peptide (via formation of a disulfide bond).

  Activated oligomers containing the above hindered esters can be obtained by methods known in the art, particularly in PCT Publication No. WO2008 / 034122 and examples thereof, which are incorporated herein by reference in their entirety. It can be synthesized by the disclosed method.

  In yet other embodiments, the oligomer of the present invention is a functionalizing reagent substantially as described in U.S. Pat. Or introducing oligomers of sulfhydryl groups, amino groups, or hydroxyl groups by a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposite end containing a hydroxyl group Is functionalized by Such reagents mainly react with oligomeric hydroxyl groups. In some embodiments, such activated oligomers have a functionalizing reagent attached to the 5′-hydroxyl group of the oligomer. In other embodiments, the activated oligomer has a functionalizing reagent attached to a 3′-hydroxyl group. In yet another embodiment, the activated oligomer of the invention has a functionalizing reagent attached to a hydroxyl group on the oligomer backbone. In further embodiments, the oligomers of the invention are functionalized with two or more of the functionalizing reagents as described in US Pat. Nos. 4,962,029 and 4,914,210, which are incorporated herein by reference in their entirety. . Methods for synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in US Pat. Nos. 4,962,029 and 4,914,210.

  In some embodiments, the 5 ′ end of the oligomer bound to the solid phase is functionalized with a dienyl phosphoramidite derivative, followed by a Diels-Alder cycloaddition reaction to convert the deprotected oligomer, for example, an amino acid or Conjugate with peptide.

  In various embodiments, incorporation of a 2'-sugar modification, eg, a monomer containing a 2'-carbamate substituted sugar or 2 '-(O-pentyl-N-phthalimido) deoxyribose sugar, into an oligomer is a conjugate. Facilitates covalent attachment of the gated moiety to the oligomeric sugar. In other embodiments, the oligomer having an amino-containing linker at the 2'-position of one or more monomers is, for example, 5'-dimethoxytrityl-2'-O- (e-phthalimidylaminopentyl) -2'- Prepared using reagents such as deoxyadenosine-3 '-N, N-diisopropyl-cyanoethoxyphosphoramidite. See, for example, Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.

  In a further embodiment, the oligomer of the present invention may have an amine-containing functional moiety at the nucleobase, such as the N6 purine amino group, the exocyclic N2 of guanine, or the N4 or 5 position of cytosine. In various embodiments, such functionalization can be achieved by using commercially available reagents that are already functionalized in oligomer synthesis.

  Several functional moiety are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from Pierce Co. (Rockford, Ill.). Other commercially available linking groups are 5'-Amino-Modifier C6 reagent and 3'-Amino-Modifier reagent, both available from Glen Research Corporation (Sterling, Va.). 5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) As Aminolink-2, and 3'-Amino-Modifier is available from Clontech Laboratories Inc. (Palo Alto, Inc. (Calib.).

Methods of Synthesis and Production The present invention also provides methods of synthesis or production of the oligomers of the present invention. Oligomers can be made using standard oligonucleotide synthesis, typically performed on a solid support, eg, a universal support. As shown in FIGS. 5 to 10, the oligomer of the present invention, for example, sequentially synthesizes the first region and the second region, and then optionally connects the third region (X) via a linker (Y). It can be synthesized by addition (eg, conjugation). Region Y (if present) may be joined to region B and then region X may be added to region Y, or region Y and region X may be added to region B in a single reaction step. Good.

  Alternatively, oligomer synthesis may be performed via first binding of region X or regions X and Y to an oligonucleotide support column followed by sequential oligonucleotide synthesis of region B and then region A.

  Alternatively, the use of a cleavable bi-directional group attached to the oligonucleotide synthesis support (in the first step or previous step) can synthesize oligonucleotide regions B and A with one reactive group of the bifunctional group. , Allowing the method of synthesizing region X or regions X and Y with a bifunctional second reactive group, where the oligonucleotide synthesis or addition of X (or X and Y) to the support is in any order Or may be performed together. Cleavage of the bifunctional group from the support then results in the oligomer of the present invention. A bifunctional group, for example, has one entity (eg, region B or X or XY-) attached to a phosphate-containing group (eg, a 5 ′ or 3 ′ group) on the nucleoside and the other (eg, Region B or X or XY-) can be, for example, a nucleoside attached to a reactive group present on a nucleobase.

  Alternatively, region X or XY may be joined to the oligomer (region B) after oligonucleotide synthesis, eg after a cleavage step. Accordingly, the present invention also relates to intermediate oligomers comprising regions A and B and reactive or activating groups attached to region B that are subsequently used to join region X or regions X and Y to region B. Related.

  Region Y or region X can be linked to region B, for example, as a phosphoramidite that allows formation of an oligomer in a single oligonucleotide synthesis followed by cleavage of the oligomer from an oligonucleotide synthesis support (US). . In this regard, in some embodiments, the linking group between region B and region X or Y is a phosphate-containing group, such as a nucleoside linkage, such as a phosphodiester, phosphorothioate, phosphodithioate, boranophosphate, methyl It can be a phosphonate, or others such as those mentioned herein. Alternatively, other chemical linkages such as triazole groups may be used.

  In some embodiments, the third region (X) or XY- is via a group other than the 5 ′ or 3 ′ phosphate, eg, a reactive group at another position, eg, a nucleoside base in region B. It can be linked to region B via the above reactive group, for example an amine.

  Oligonucleotide synthesis may be performed in the 5 ′ to 3 ′ direction, or may be performed in the 3 ′ to 5 ′ direction, as is typical for most oligonucleotide synthesis.

（B）構築物のB〜A成分を、ホスホロチオエート連結およびホスホジエステル連結の両方を合成することができるオリゴヌクレオチド合成機で作製することができる。次いで、任意で、X成分とA成分との間にPO連結またはPS連結を有するX-A-B-Aを作製するため、ビルディングブロックDMTrO-A-Pを使用し、続いて、ビルディングブロックX-Pを使用して、標準的なホスホラミダイト化学によって、B〜Aを逐次延長することができる。

In some non-limiting examples, the oligonucleotide-conjugate construct can be assembled in different ways. For example,
(A) The B-A component of the construct can be made on an oligonucleotide synthesizer capable of synthesizing both phosphorothioate and phosphodiester linkages. Then, optionally, using building block XAP (conjugate moiety with linker attached) to make XABA, or using building block XP (conjugate part without linker) to make XBA B to A can be extended by standard phosphoramidite chemistry.

(B) The B-A component of the construct can be made on an oligonucleotide synthesizer capable of synthesizing both phosphorothioate and phosphodiester linkages. Then, optionally, using the building block DMTrO-AP, followed by the building block XP to create a XABA with PO or PS linkage between the X and A components, B to A can be extended sequentially by phosphoramidite chemistry.

The B-A component of the construct can be made on an oligonucleotide synthesizer that can synthesize both phosphorothioate and phosphodiester linkages. Then, optionally, for producing H ₂ NABA, it may use the building blocks PGN-AP, successively extending the B~A by standard phosphoramidite chemistry. After cleavage and deprotection of the oligonucleotide, the free amine of the oligonucleotide can be conjugated with the moiety X activated so that the functional group of X reacts with the terminal primary amine of the oligonucleotide.

Compositions The oligomers of the invention may be used in pharmaceutical formulations and compositions. Where appropriate, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt, or adjuvant. WO2007 / 031091 provides suitable and preferred pharmaceutically acceptable diluents, carriers and adjuvants, which are incorporated herein by reference. Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO2007 / 031091, which are also incorporated herein by reference.

  Antisense oligonucleotides can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

  Antisense compounds can be utilized in pharmaceutical compositions by combining the antisense compounds with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS). PBS is a suitable diluent for use in compositions that are delivered parenterally.

  A pharmaceutical composition comprising an antisense compound comprises a pharmaceutically acceptable salt, ester, or salt of such ester, or a metabolite or residue thereof that has biological activity upon administration to an animal, including a human. Other oligonucleotides that can provide (directly or indirectly). Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents of antisense compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. Prodrugs can include the incorporation of additional nucleosides that are cleaved by endogenous nucleases in the body to form an active antisense compound at one or both ends of the antisense compound. In this regard, the prodrug may comprise region B according to the present invention and a conjugate moiety, targeting moiety or blocking moiety. In some embodiments, the oligomer of the invention is a prodrug.

  The use of lipophilic conjugates according to the present invention can be used, for example, in lipidoids or liposomes that are particularly useful for delivery of oligomers to the liver, eg siRNA, eg cationic liposomes, eg cationic liposome SNALP ( Enables incorporation of the oligomers of the invention into stable nucleic acid lipid particles).

Applications The oligomers of the present invention can be utilized, for example, as research reagents for diagnosis, treatment, and prevention.

  In research, in some embodiments, such oligomers are responsible for protein synthesis in cells and laboratory animals (typically by degrading or inhibiting mRNA, thereby preventing protein formation). It can be used to specifically inhibit, thereby facilitating functional analysis of the target, or evaluation of its usefulness as a target for therapeutic intervention.

  For therapy, an animal or human suspected of having a disease or disorder that can be treated by modulating the expression of the target is treated by administering an oligomeric compound according to the invention. A method of treating a mammal by administering one or more of the oligomers or compositions of the invention in a therapeutically or prophylactically effective amount, eg, a disease or condition associated with target expression. Further provided are methods of treating a human having or suspected of having a predisposition thereto. The oligomer, conjugate or pharmaceutical composition according to the present invention is typically administered in an effective amount.

  The invention relates to a compound or conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder referred to herein or for the method of treatment of a disorder referred to herein. It also provides the use of gates.

  The present invention comprises administering a compound according to the invention described herein and / or a conjugate according to the invention and / or a pharmaceutical composition according to the invention to a patient in need thereof. Also provided are methods of treating the disorders mentioned in the specification.

Medical indications In some embodiments, the disease is cancer. In some embodiments, the disease is an inflammatory disease. In some embodiments, the disease is a cardiovascular disease, and in some embodiments, the disease or disorder is myocardial infarction (MI).

  In some embodiments, the disease or disorder is fibrosis, eg, liver fibrosis, cardiac fibrosis, or local fibrosis, or causes or is associated with them.

  In some embodiments, the disease or disorder is a blood clotting disorder.

  In some embodiments, the disease or disorder is or includes (causes or is associated with) bone loss.

  In some embodiments, the disease or disorder is a liver disease or disorder.

  In some embodiments, the disease or disorder is a metabolic disorder that can be, for example, a liver disease or disorder, and / or in some aspects a cardiovascular disease or neovascular disorder.

  Cardiovascular / metabolic disorders include, for example, metabolic syndrome, obesity, hyperlipidemia, HDL / LDL cholesterol imbalance, dyslipidemia such as familial combined hyperlipidemia (FCHL), acquired hyperlipidemia , Statin resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD), atherosclerosis, heart disease, diabetes (type I and / or type II), NASH, acute coronary syndrome ( ACS), NASH, chronic heart failure, heart disease, cardiometabolic disease, hyperlipidemia and related disorders, metabolic syndrome, atherosclerosis, chronic heart failure, vascular disease, peripheral arterial disease, heart disease, ischemia, type 2 Diabetes and type 1 diabetes are included.

  In some embodiments, the disease or disorder is metabolic syndrome, obesity, hyperlipidemia, atherosclerosis, HDL / LDL cholesterol imbalance, dyslipidemia, eg, familial combined hyperlipidemia (FCHL ), Acquired hyperlipidemia, statin resistant hypercholesterolemia, coronary artery disease (CAD), and coronary heart disease (CHD).

  In some embodiments, the disease or disorder is selected from the group consisting of chronic heart failure, cardiovascular disease, cardiac metabolic disease, chronic heart failure, vascular disease, peripheral arterial injury, heart disease, ischemia, acute coronary syndrome (ACS). The

  In some embodiments, the disease or disorder is type 2 diabetes, type 1 diabetes.

  In some embodiments, the disease or disorder is a viral disease such as erythrocytosis, hepatitis C, hepatitis B, BKV, HIV.

  In some embodiments, the disease or disorder is a serious rare disease (or genetic disease).

  The present invention further provides the use of a compound of the present invention in the manufacture of a medicament for the treatment of a disease, disorder or condition, eg, those mentioned herein.

  Generally speaking, some aspects of the invention include a target comprising administering to a mammal a therapeutically effective amount of an oligomer targeted to a target comprising one or more LNA units. It relates to a method of treating a mammal suffering from or susceptible to a condition associated with an abnormal level of. The oligomer, conjugate or pharmaceutical composition according to the present invention is typically administered in an effective amount.

  An important aspect of the present invention relates to the use of a compound as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition referred to herein.

  Furthermore, the present invention relates to a method of treating a subject suffering from a disease or condition, eg, those mentioned herein.

  A patient in need of treatment is a patient suffering from or likely to have a disease or disorder.

  In some embodiments, as used herein, the term “treatment” refers to the treatment of an existing disease (eg, a disease or disorder referred to herein) or the prevention, ie, prevention of a disease. Point to both. Accordingly, it will be appreciated that the treatments referred to herein may be prophylactic in some embodiments.

List of oligonucleotides In the following list, upper case letters represent LNA nucleosides, such as β-D-oxy LNA, and lower case letters represent DNA nucleosides. The uppercase L is LNA, such as β-D-oxy, and the lowercase d is a DNA nucleoside. The LNA cytosine may optionally be 5 ′ methyl cytosine. The internucleoside linkage in region A is a phosphorothioate, and the internucleoside linkage in region B is a phosphodiester (as indicated). The internucleoside linkage between regions A and B is a phosphodiester, but if region B is a DNA nucleotide of> 1, it may optionally be other than a phosphodiester (e.g., phosphorothioate). ). There may optionally be an additional linker (Y) between region B and region C, such as a C6 linker. # Means SEQ ID No.

SEQ ID NO 53はSEQ ID NO 54の親化合物として提供されている。 In the above compound, region C has a complement to Seq (region A) so that the 3 ′ nucleotide of region C is aligned (base paired) with the 8th nucleotide of region A from the 5 ′ end. Including. Region C is therefore bent and forms an 8 base hybridization with region A over the 3 ′ wing of the gapmer and 5 bases of the DNA gap region, thereby inactivating until the linker region (B) is cleaved A “prodrug” is produced.

SEQ ID NO 53 is provided as the parent compound of SEQ ID NO 54.

Mouse tests: Unless otherwise specified, mouse experiments can be performed as follows:
Dosage administration and sampling:
Seven to ten week old C57Bl6-N mice were used and the animals were matched for age and gender (female in tests 1, 2 and 4 and male in test 3). The compound was injected intravenously into the tail vein. For intermediate serum sampling, 2-3 drops of blood were collected by puncture of the facial vein and the final blood was taken from the inferior vena cava. Serum was collected in a serum separator tube (Greiner) containing the gel and stored frozen until analysis.

  C57BL6 mice were dosed intravenously with a single dose (or indicated amount) of 1 mg / kg ASO formulated in saline according to the indicated information or saline alone. The animals were sacrificed, for example on day 4 or 7 (or when indicated) after dosing and liver and kidney were sampled.

RNA isolation and mRNA analysis:
Tissue-derived mRNA analysis was performed using the Qantigene mRNA quantification kit ("bDNA-assay", Panomics / Affimetrix) according to the manufacturer's protocol. In the case of tissue lysates, 50-80 mg of tissue was lysed by sonication in 1 ml of lysis buffer containing proteinase K. Lysates were used directly for bDNA-assay without RNA extraction. Probe sets for target and GAPDH were custom designed and obtained from Panomics. For analysis, the luminescence units obtained for the target gene were normalized to the housekeeper GAPDH.

  Serum analysis of ALT, AST and cholesterol was performed on the “Cobas INTEGRA 400 plus” clinical chemistry platform (Roche Diagnostics) using 10 μl of serum.

  For quantification of serum levels of Factor VII, the BIOPHEN FVII enzyme activity kit (# 221304, Hyphen BioMed) was used according to the manufacturer's protocol.

  For oligonucleotide quantification, a fluorescently labeled PNA probe is hybridized with the oligo of interest in the tissue lysate. For the bDNA-assay method, use the same lysate with only a precisely weighed amount of tissue. Heteroduplexes are quantified using AEX-HPLC and fluorescence detection.

Example 1: Synthesis of compounds SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5 They were synthesized using the phosphoramidite method on the Multiple Oligonucleotide Synthesis System) or the equivalent on a 4 μmol scale. At the end of the synthesis, the oligonucleotide was cleaved from the solid support with aqueous ammonia for 1-2 hours at room temperature and further deprotected at 65 ° C. for 16 hours. Oligonucleotides were purified by reverse phase HPLC (RP-HPLC), characterized by UPLC, and molecular weight was further confirmed by ESI-MS. See below for more details.

Oligonucleotide extension β-cyanoethyl-phosphoramidite (DNA-A (Bz), DNA-G (ibu), DNA-C (Bz), DNA-T, LNA-5-methyl-C (Bz), LNA- (A (Bz), LNA-G (dmf), LNA-T or C6-SS linker) is bound to 0.1 M of 5'-O-DMT protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole). Acetonitrile solution (0.25 M) is used as the activator. For the last cycle, a commercially available C6-linked cholesterol phosphoramidite was used at 0.1 M in DCM. Thiolation for the introduction of phosphorothioate linkages is performed by using xanthan hydride (0.01 M in acetonitrile / pyridine 9: 1). The phosphodiester bond is introduced using 0.02 M iodine in THF / pyridine / water 7: 2: 1. The rest of the reagents are those normally used for oligonucleotide synthesis.

Purification by RP-HPLC:
The crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150 × 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile were used as buffer at a flow rate of 5 mL / min. The collected fractions were lyophilized to give the purified compound, typically as a white solid.

Abbreviations:
DCI: 4,5-dicyanoimidazole
DCM: dichloromethane
DMF: Dimethylformamide
DMT: 4,4'-dimethoxytrityl
THF: tetrahydrofuran
Bz: Benzoyl
Ibu: Isobutyryl
RP-HPLC: Reversed phase high performance liquid chromatography

Example 2: LNA antisense oligonucleotide design Oligomers used in the examples and figures. SEQ # is an identifier used throughout the examples and figures.

Example 3 Apo B mRNA knockdown by cholesterol conjugates in vivo
C57BL6 / J mice are conjugated to cholesterol with a single dose of saline or 1 mg / kg unconjugated LNA-antisense oligonucleotide (SEQ ID # 1), or equimolar amounts of various linkers. LNA antisense oligonucleotides were injected and sacrificed on days 1-10 according to Table 3.

  RNA was isolated from liver and kidney and subjected to qPCR with apo B specific primers and probes to analyze for apo B mRNA knockdown.

Conclusion:
Cholesterol (Seq # 4 and 5) conjugated to apoB LNA antisense oligonucleotides with linkers composed of 2 or 3 DNAs with a phosphodiester backbone is a liver-specific knockout of apoB Down priority was shown (Figure 11). Compared to the unconjugated compound (Seq # 1) and the cholesterol conjugate with a stable linker (Seq # 2) and the cholesterol conjugate with a disulfide linker (Seq. # 3) This means that the efficiency and duration of apo B mRNA knockdown is increased, and at the same time, the knockdown activity of Seq # 4 and # 5 in kidney tissue is lower.

Materials and methods:
Experimental design:

Dose administration Approximately 20 g of C57BL / 6JBom female animals upon arrival are intravenously administered with compound or saline only formulated in saline according to Table 3 at 10 ml / kg body weight (according to day 0 body weight). It was administered internally.

Sampling Liver and Kidney Tissue Animals were anesthetized with 70% CO ₂ -30% O ₂ and sacrificed by cervical dislocation according to Table 3. Half the large liver lobe and one kidney were minced and immersed in RNAlater.

Total RNA isolation and first strand synthesis
Total RNA was extracted from a maximum of 30 mg of tissue homogenized by bead milling in the presence of RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen catalog number 74106) according to the manufacturer's instructions. First strand synthesis was performed using Ambion's reverse transcriptase reagent according to the manufacturer's instructions.

For each sample, adjust 0.5 μg of total RNA to 10.8 μl with RNase-free H ₂ O, mix with 2 μl of random decamer (50 μM) and 4 μl of dNTP mix (2.5 mM each dNTP), 70 Heated to 3 ° C. for 3 minutes, after which the sample was quickly cooled on ice. Add 10 μl buffer RT 2 μl, MMLV reverse transcriptase (100 U / μl) 1 μl and RNase inhibitor (10 U / μl) 0.25 μl to each sample, followed by incubation at 42 ° C for 60 minutes, at 95 ° C The enzyme was heat inactivated for 10 minutes, after which the sample was cooled to 4 ° C. RT-QPCR using a Taqman Fast Universal PCR Master Mix 2X (Applied Biosystems Cat # 4364103) and Taqman gene expression assays (mApo B, Mn01545150_m1 and mGAPDH # 4352339E) according to the manufacturer's protocol And processed in high speed mode on an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7).

Example 4 In vivo knockdown of apo B mRNA by cholesterol conjugate and LNA distribution to liver and kidney
C57BL6 / J mice are conjugated to cholesterol with a single dose of saline or 1 mg / kg unconjugated LNA-antisense oligonucleotide (SEQ ID # 1), or equimolar amounts of various linkers. LNA antisense oligonucleotides were injected and sacrificed on days 1-16 according to Table 4. RNA was isolated from liver and kidney and subjected to qPCR with apo B specific primers and probes to analyze for apo B mRNA knockdown. LNA oligonucleotide content was measured in liver and kidney using a sandwich ELISA method based on LNA.

Conclusion:
Cholesterol (Seq # 4, # 5, and # 6) conjugated to an apoB LNA antisense oligonucleotide with a linker composed of 1, 2, or 3 DNA with a phosphodiester backbone is The liver-specific knockdown preference of Apo B was shown (FIG. 14). This is due to the increased efficiency and duration of apo B mRNA knockdown in liver tissue compared to unconjugated compound (Seq # 1), and at the same time, Seq # 4, # 5, and # 6 means lower knockdown activity. Cholesterol-conjugated LNA antisense oligonucleotides have higher uptake in the liver and lower uptake in the kidney compared to unconjugated LNA oligonucleotides (FIG. 15).

Materials and methods:
Experimental design:

Dose administration Approximately 20 g of C57BL / 6JBom female animals upon arrival are intravenously administered with a compound formulated in saline according to Table 4 or saline alone at 10 ml / kg body weight (according to body weight on day 0). It was administered internally.

Sampling Liver and Kidney Tissue Animals were anesthetized with 70% CO ₂ -30% O ₂ and sacrificed by cervical dislocation according to Table 4. Half the large liver lobe and one kidney were minced and immersed in RNAlater.

Total RNA isolation and first strand synthesis
Total RNA was extracted from a maximum of 30 mg of tissue homogenized by bead milling in the presence of RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen catalog number 74106) according to the manufacturer's instructions.

  First strand synthesis was performed using Ambion's reverse transcriptase reagent according to the manufacturer's instructions.

For each sample, adjust 0.5 μg of total RNA to 10.8 μl with RNase-free H ₂ O, mix with 2 μl of random decamer (50 μM) and 4 μl of dNTP mix (2.5 mM each dNTP), 70 Heated to 3 ° C. for 3 minutes, after which the sample was quickly cooled on ice. Add 10 μl buffer RT 2 μl, MMLV reverse transcriptase (100 U / μl) 1 μl and RNase inhibitor (10 U / μl) 0.25 μl to each sample, followed by incubation at 42 ° C for 60 minutes, at 95 ° C The enzyme was heat inactivated for 10 minutes, after which the sample was cooled to 4 ° C. RT-QPCR using a Taqman Fast Universal PCR Master Mix 2X (Applied Biosystems Cat # 4364103) and Taqman gene expression assays (mApo B, Mn01545150_m1 and mGAPDH # 4352339E) according to the manufacturer's protocol And processed in high speed mode on an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7).

Oligo content sandwich ELISA:
Liver and kidney samples (100 mg) were collected in test tubes at various times after dosing. Add buffer (pH 8.0 100 mM NaCl, 25 mM EDTA, 0.25 mM Tris), protease k (1%, Sigma P4850-5) and 2 Tungsten Carbide Beads (3 mm) (Qiagen) to the sample and homogenize for 8 minutes. (Retsch MM300, 25 Hz [l / s]), the homogenate was incubated overnight at 37 ° C. Samples were spun at 14000 g for 15 minutes before use.

  LNA oligonucleotide standards 1-100 μg / g in kidney and liver were prepared and processed as described above. Standards and samples were added to a 35 nM solution of biotinylated and digoxigenin-modified capture and detection probes (5 × SSCT buffer [(750 mM NaCl, and 75 mM sodium citrate, 0.05% (v / v) Tween -20 pH 7.0)] was diluted to 150 μl (100-5000 ng / L) and mixed for 1 hour.The streptavidin coating (Nunc Immobilizer Streptavidin F96 CLEAR module plate Nunc catalog number 436014) was washed 3 times (5 × SSCT buffer, 300 μl) 100 μL of sample was transferred to streptavidin-coated plate and incubated for 1 hour under gentle shaking.Wells were aspirated and 2 × SSCT buffer (0.05% (v / v ) Washed 3 times with 300 μl of 300 mM NaCl + 30 mM sodium citrate containing Tween-20, pH 7.0 Anti-Dig- diluted 1: 4000 in PBST (phosphate buffered saline, pH 7.2) AP Fab fragment (Roche Applied Science, catalog number 11 093 274 910) 100 My The liters were added to the wells and incubated for 1 hour at room temperature with gentle agitation The wells were aspirated and washed 3 times with 300 μl of 2 × SSCT buffer Substrate solution (KPL BluePhos Microwell Phosphatase substrate system 100 microliters was added to each well, and the intensity of color development was measured spectrophotometrically at 615 nm every 5 minutes after shaking.

Example 5: Knockdown of PCSK9 mRNA by cholesterol conjugate in vivo
NMRI mice are given a single dose of saline, or 10 mg / kg unconjugated LNA-antisense oligonucleotide (SEQ ID 7), or equimolar amounts of LNA antigen conjugated to cholesterol with various linkers. Sense oligonucleotides were injected and were sacrificed on days 1-10 according to Table 5.

  RNA was isolated from liver and kidney and subjected to qPCR with PCSK9-specific primers and probes to analyze for PCSK9 mRNA knockdown.

Conclusion:
Cholesterol (Seq # 9 and # 10) conjugated to a PCSK9 LNA antisense oligonucleotide with a linker composed of two DNAs with a phosphodiester backbone is compared to an unconjugated compound (Seq # 7) And enhanced liver knockdown of PCSK9 (FIG. 16) compared to cholesterol conjugate with stable linker (Seq # 8).

Materials and methods:
Experimental design:

Dose Administration Approximately 20 g of NMRI female animals upon arrival, intravenously administered with compound or saline only formulated in saline according to Table 5 at 10 ml / kg body weight (according to day 0 body weight) did.

Sampling Liver and Kidney Tissue Animals were anesthetized with 70% CO ₂ -30% O ₂ and sacrificed by cervical dislocation according to Table 4. Half the large liver lobe and one kidney were minced and immersed in RNAlater.

Homogenized by bead milling in the presence of MagNA Pure LC RNA Isolation Tissue buffer (Roche catalog number 03 604 721 001) using MagNa Pure 96 Cellular RNA Large Volume kit (Roche catalog number 5467535001) according to manufacturer's instructions Total RNA was extracted from up to 10 mg of tissue. First strand synthesis was performed using Ambion's reverse transcriptase reagent according to the manufacturer's instructions. For each sample, adjust 0.5 μg of total RNA to 10.8 μl with RNase-free H ₂ O, mix with 2 μl of random decamer (50 μM) and 4 μl of dNTP mix (2.5 mM each dNTP), 70 Heated to 3 ° C. for 3 minutes, after which the sample was quickly cooled on ice. Add 10 μl buffer RT 2 μl, MMLV reverse transcriptase (100 U / μl) 1 μl and RNase inhibitor (10 U / μl) 0.25 μl to each sample, followed by incubation at 42 ° C for 60 minutes, at 95 ° C The enzyme was heat inactivated for 10 minutes, after which the sample was cooled to 4 ° C. Dilute the cDNA sample 1: 5 and perform RT-QPCR using Taqman Fast Universal PCR Master Mix 2X (Applied Biosystems Cat # 4364103) and Taqman gene expression assay (mPCSK9, Mn00463738_m1 and m actin # 4352341E) according to the manufacturer's protocol And processed in high speed mode on an Applied Biosystems RT-qPCR instrument (7500/7900 or ViiA7).

Example 6. In vitro cleavage of various DNA / PO-linkers FAM-labeled ASO with various DNA / PO-linkers (PO linkers) was subjected to in vitro cleavage in S1 nuclease extract (Figure 6A), liver or kidney 100 μM FAM-labeled ASO with various DNA PO-linkers in homogenate or serum was subjected to in vitro cleavage with S1 nuclease (60 U / 100 μL) in nuclease buffer for 20 and 120 minutes (A). Enzymatic activity was stopped by adding EDTA to the buffer solution. The solution was then subjected to AIE HPLC analysis on a Dionex Ultimate 3000 using a Dionex DNApac p-100 column and a gradient ranging from 10 mM to 1 M sodium perchlorate pH 7.5. Using a fluorescence detector at 615 nm and a uv detector at 260 nm, the content of cleaved and uncleaved oligonucleotides was determined relative to standards.

Conclusion:
The PO linker (or region B as referred to herein) allows the conjugate (or group C) to be cleaved, using both the linker length and / or sequence composition, region B Can be sensitive to nucleolytic cleavage. As seen after 20 minutes in the nuclease S1 extract, the sequence of the DNA PO-linker can regulate the rate of cleavage, and therefore using sequence selection for region B (eg for the DNA PO-linker) It is also possible to regulate the level of cleavage in serum and in cells of the target tissue.

Oligonucleotide SEQ ID NO 16 was added to liver, kidney, and serum (B) to a concentration of 200 μg / g tissue. Liver and kidney samples collected from NMRI mice were homogenized in homogenization buffer (0,5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjusted with 1 N NaOH)). The homogenate was incubated for 24 hours at 37 ° C., after which the homogenate was extracted with phenol / chloroform.The contents of cleaved and uncleaved oligonucleotides in extracts from liver and kidney, and from serum were determined by HPLC as described above. The standard substance was determined using the method.

Conclusion:
The PO linker (or region B as referred to herein) results in cleavage of the conjugate (or group C) from the oligonucleotide in liver or kidney homogenate, but not in serum.

Notes:
Cleavage in the above assay refers to cleavage of the cleavable linker and the oligomer or region A must remain functionally complete. Sensitivity to cleavage in the above assay can be used to determine whether the linker is biologically cleavable or physiologically unstable.

Example 7a: In vivo inhibition of FVII (1 mg / kg)
An in vivo mouse study was prepared using a total of 6 groups of mice (n = 3). Each mouse received a single intravenous dose of LNA compound targeting FVII mRNA at 1 mg / kg or equimolar compared to SEQ ID # 12. A saline control group was included. One day before the administration, blood was collected from the mouse in advance, and the subsequent blood collection was performed on the first and second days after the administration. Mice were sacrificed on day 4 and liver, kidney, and blood were collected. See Table 7 for test settings.

  Standard assay techniques were used to measure factor VII serum levels, mRNA levels, and oligonucleotide tissue content.

Conclusion:
DNA PO-linker (PO) improves the downregulation of serum FVII protein for FVII mRNA targeted LNA oligonucleotides with cholesterol conjugates when compared to the widely used dithiolinker (disulfide) (Fig. 18) (PO linker SEQ ID # 15 is comparable to SS linker SEQ ID # 14). When using GalNAc as a conjugate, it is clear that the PO linker improves the down-regulation of the FVII protein compared to the amino-linked conjugate LNA oligonucleotide (PO linker SEQ ID # 13 is the amino-linked SEQ. Equivalent to ID # 12). GalNac conjugates are known to be biologically cleavable (possibly by a peptide linker), so PO linkers further enhance the release of active and potent compounds in target cells. I think that the. These data are consistent with the mRNA expression data (Figure 19). The tissue content of oligonucleotides in the kidney and liver shows how the conjugate changes its distribution (Figure 20). The two cholesterol conjugate compounds show a similar distribution (compare SEQ ID # 14 and # 15), thus showing enhanced mRNA and FVII protein downregulation of the PO linker compound (SEQ ID # 15) Shows how the activity of LNA oligonucleotides targeting FVII is enhanced compared to SEQ ID # 14.

Materials and methods:
Experimental design:

  Female mice were administered intravenously and liver, kidney and blood were sampled at day 4 sacrifice. Additional blood collection was performed before dosing and on days 1 and 2 after dosing.

Example 7b: In vivo inhibition of FVII (0,1 and 0,25 mg / kg)
A total of 7 groups of mice (n = 3) were used for in vivo mouse testing. Each mouse received a single intravenous dose of an LNA compound targeting FVII mRNA at either an equimolar amount of 0,1 mg / kg or 0,25 mg / kg compared to SEQ ID # 12 . A saline control group was included. One day before administration, blood was collected from the mouse in advance, and subsequent blood collection was performed on the 4th, 7th, 11th, 14th, and 18th days after administration. Mice were sacrificed on day 24 and liver, kidney, and blood samples were collected. See Table 8 for test settings. Standard assay techniques were used to measure factor VII serum levels, mRNA levels, and oligonucleotide tissue content.

Conclusion:
DNA / PO-linker (PO) at both 0,1 mg / kg and 0.25 mg / kg, FVII mRNA targeted LNA oligonucleotide with cholesterol conjugate when compared to the widely used dithiolinker Improve the down-regulation of FVII protein (FIG. 21) (PO linker SEQ ID # 15 is comparable to SS linker SEQ ID # 14). When using GalNAc as a conjugate, mRNA data (Figure 22) suggests that PO linkers improve downregulation compared to amino-linked conjugate LNA oligonucleotides (PO linker SEQ ID # 13 is Equivalent to amino-linked SEQ ID # 12). The mRNA expression data (FIG. 22) support that the improved activity of the PO linker compound (SEQ ID # 15) is comparable to the dithio-linked conjugate (SEQ ID # 14). The tissue content of oligonucleotides in the kidney and liver shows how the conjugate changes its distribution (Figure 23). The data suggests that PO linkers enhance uptake in both liver and kidney at these dose ranges relative to both cholesterol and GalNAc conjugates (compare SEQ ID # 14 and # 15, and Compare SEQ ID # 13 with # 12).

Materials and methods:
Experimental design:

  Male mice were administered intravenously, and liver, kidney, and blood were sampled at day 24 of sacrifice. Additional blood collections were performed prior to dosing and on days 4, 7, 11, 14, and 18 after dosing.

Example 8. In vivo silencing of apoB mRNA with various conjugates and PO-linkers To explore the effects of biocleavable DNA PO-linkers on additional conjugates, C57BL6I mice were treated with saline control or simply A single dose of 1 mg / kg of parent compound # 1, or an equimolar amount of mono-GalNAc, folic acid, without biocleavable linker, with dithio-linker (SS), or with DNA / PO-linker (PO), Intravenous treatment with Fam or ASO conjugated to tocopherol. Seven days later, the animals were sacrificed and RNA was isolated from liver and kidney samples and analyzed for apo B mRNA expression (Figure 24).

Conclusion:
For all four conjugates, the DNA / PO-linker improves apo B knockdown in the liver compared to the widely used dithio-linker (# 27 is # 26, # 30 is # 29, Compare # 33 with # 32 and # 36 with # 35). In the case of mono-GalNAc and tocopherol, the DNA / PO-linker improves knockdown of apo B in the liver, even compared to unconjugated compounds (compare # 30 and # 36 with # 1). Tocopherol in combination with a DNA / PO-linker shows the ability to redirect the compound from the kidney to the liver (compare A and B, # 36 with # 1).

Materials and methods:
Experimental design:

Dosage administration and sampling
C57BL6 mice were intravenously dosed with a single dose of 1 mg / kg ASO formulated in saline according to the table above or saline alone. Animals were sacrificed 7 days after dosing and liver and kidney were sampled.

RNA isolation and mRNA analysis Total RNA was extracted from liver and kidney samples and analyzed for apo B mRNA levels using a branched DNA assay.

Example 9. In vitro silencing of target X mRNA by loop LNA ASO with PO-linker Blocker groups may be beneficial in terms of ASO tolerance, specificity or reduced off-target effects, but the original, This can be difficult in terms of preserving the activity of unblocked ASO. As an example of a blocker group, we used a complementary sequence linked to the oligonucleotide by a non-complementary nucleotide stretch that produced a hairpin loop. The unpaired base in the loop was either three DNA nucleotides with a phosphodiester backbone (PO-linker) or the same DNA nucleotide with a phosphorothioate backbone. To test the activity of loop LNA-ASO, Neuro 2a cells were treated with 1 μM ASO in a Jimnosis assay, RNA extracted, and subjected to RT-QPCR to analyze for target X mRNA knockdown (Figure 25). ).

Conclusion:
Loop ASO with PO-linker (Seq ID # 21 and # 23) shows improved knockdown of target X mRNA compared to the same ASO sequence without PO-linker (Seq ID # 22 and # 24) It was.

Materials and methods:
Gymnosis assay in N2a cells:
Neuro 2a (mouse neuroblastoma) cells were seeded in a 24-well plate at 1.8 × 10 ⁴ cells / well, DMEM + Glutamax (gibco-life, # 61965-026), 2 mM glutamine, 10% FBS, 1 Treated with 1 μM loop LNA ASO with and without PO-linker in mM sodium pyruvate, 25 μg / ml gentamicin, respectively.

Total RNA isolation and first strand synthesis
Total RNA was extracted 6 days after Jimnosis using the Qiagen RNeasy kit (Qiagen catalog number 74106) according to the manufacturer's instructions. First strand synthesis was performed using Ambion's reverse transcriptase reagent according to the manufacturer's instructions.

  For each sample, mix 0.5 μg of total RNA with 2 μl of random decamer (50 μM) and 4 μl of dNTP mix (2.5 mM each dNTP) and heat to 70 ° C. for 3 minutes, after which the sample is quickly squeezed on ice. Cooled down. Add 10 μl buffer RT 2 μl, MMLV reverse transcriptase (100 U / μl) 1 μl and RNase inhibitor (10 U / μl) 0.25 μl to each sample, followed by incubation at 42 ° C for 60 minutes, at 95 ° C The enzyme was heat inactivated for 10 minutes, after which the sample was cooled to 4 ° C. The cDNA sample is diluted 1: 5 and subjected to RT-QPCR using Taqman Fast Universal PCR Master Mix 2X (Applied Biosystems Cat # 4364103) and Taqman gene expression assay against target X according to the manufacturer's protocol, and Applied Biosystems RT-qPCR Processed in high-speed mode on the instrument (ViiA7). Target X mRNA expression was normalized to β-actin mRNA expression (mBACT # 4352341E) and compared to mock mRNA levels.

Example 10: Non-human primate study The main objective of this study is to examine selected lipid markers over 7 weeks after a single slow intravenous bolus of anti-PCSK9 and anti-apo B LNA conjugate compounds to cynomolgus monkeys And assessing the potential toxicity of the compound in monkeys. The compounds used in this study were SEQ ID NOs 46 and 49, 5 and 54, which were prepared in sterile saline (0.9%) at initial concentrations of 0.625 and 2.5 mg / ml.

  Use male (PCSK9) or female (ApoB) monkeys at least 24 months of age, making them free to use tap water and overfeeding MWM (E) SQC SHORT per animal (Dietex France, SDS, Saint Gratien , France) 180 g shall be distributed daily. The total amount of feed distributed to each cage is calculated by the number of animals in the cage of the day. In addition, fruits or vegetables are given to each animal daily. Animals are habituated to the test conditions for at least 14 days prior to the start of the treatment period. During this period, a pretreatment study will be conducted. Animals are administered intravenously at one dose, for example, at 0.25 mg / kg or 1 mg / kg. The dose is 0.4 mL / kg. Use 2 animals per group. After 3 weeks, data can be analyzed and a second dose group can be undertaken using higher or lower dose schedules-preliminary dose settings are 0.5 mg / kg and 1 mg / kg Or lower than that based on the first data set.

  The dose formulation is administered once a day. Animals are observed for 7 weeks after treatment and released from the study on day 51. The first day is the first day of the treatment period. Clinical observations and body weight and food intake (per group) are recorded before and during the study.

RCP 0 日常的な臨床病理学、LSB = 肝臓安全性の生化学、PK = 薬物動態学、OA = 他の分析、L = 脂質。 Blood is sampled and analyzed at the following times:

RCP 0 Routine clinical pathology, LSB = Liver safety biochemistry, PK = Pharmacokinetics, OA = Other analysis, L = Lipid.

Blood biochemistry The following parameters are determined for all surviving animals when:
● Complete biochemistry panel (complete list below)-days -8, 15, and 50,
● Liver safety (ASAT, ALP, ALAT, TBIL and GGT only)-Day 4, 8, 22, and 36,
● Lipid profile (total cholesterol, HDL-C, LDL-C, and triglycerides) and Apo-B only-days 1, 4, 8, 22, 29, 36, and 43.

  Take blood (approximately 1.0 mL) into a lithium heparin tube (using ADVIA 1650 blood biochemical analyzer): Apo-B, sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, Urea, creatinine, total bilirubin (TBIL), total cholesterol, triglycerides, alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), creatine kinase, γ-glutamyltransferase (GGT), lactate dehydrogenation Enzyme, total protein, albumin, albumin / globulin ratio.

Blood analysis:
Blood samples for PCSK9 analysis are collected only from group 16 animals on days -8, -1, 4, 8, 15, 22, 29, 36, 43, and 50.

  Venous blood (approximately 2 mL) is collected from the appropriate vein in each animal into a serum separator tube (SST) and allowed to clot for at least 60 ± 30 minutes at room temperature. Blood is centrifuged at 1000 g for 10 minutes under refrigerated conditions (set to maintain + 4 ° C). Transfer serum to 3 separate tubes and store at -80 ° C until analyzed in CitoxLAB France using ELISA method (Circulex Human PCSK9 ELISA kit, CY-8079, validated against cynomolgus serum) did.

Other analysis:
WO2011009697 and WO2010142805 provide methods for the following analysis: qPCR, PCSK9 / ApoB mRNA analysis. Other analyzes include PCSK9 / Apo B protein ELISA, serum Lp (a) analysis by ELISA (Mercodia number 10-1106-01), tissue and plasma oligonucleotide analysis (drug content), sample extraction, standard samples and QC Includes sample, determination of oligonucleotide content by ELISA.

Example 11: Liver and kidney toxicity assessment in rats The compounds of the invention can be evaluated for their toxicity profile in rodents, such as in mice or rats. As an example, the following protocol may be used: Wistar Han Crl: WI (Han) is used at an age of approximately 8 weeks of age. At this age, males should weigh approximately 250 g. All animals have free access to the SSNIFF R / MH pellet maintenance diet (SSNIFF Spezialdiaten GmbH, Soest, Germany) and the tap water contained in the bottle (filtered through a 0.22 μm filter). Doses are administered on days 1 and 8 using dose levels of 10 and 40 mg / kg / dose (subcutaneous administration). Animals are euthanized on day 15. Urine and blood samples are collected on days 7 and 14. Clinicopathological assessment is performed on day 14. Body weight is determined prior to testing, on the first day of dosing, and 1 week before necropsy. Evaluate feed consumption per group daily. Blood samples are taken from the tail vein 6 hours after fasting. Perform the following serum analysis: Red blood cell count Average cell volume Blood blood cell volume Hemoglobin Average cell hemoglobin concentration Average cell hemoglobin Platelet count White blood cell count White blood cell fraction with cell morphology Reticulocyte count, sodium potassium chloride calcium inorganic phosphorus glucose urea creatinine total bilirubin Total cholesterol Triglyceride Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Total protein Albumin Albumin / globulin ratio. Urinalysis is performed for α-GST, β-2 microglobulin, calbindin, clusterin, cystatin C, KIM-1, osteopontin, TIMP-1, VEGF, and NGAL. Seven analytes (calbindin, clusterin, GST-α, KIM-1, osteopontin, TIMP-1, VEGF) are panel 1 (MILLIPLEX® MAP Rat Kidney Toxicity Magnetic Bead Panel 1, RKTX1 MAG-37K) Quantified under. Three analytes (β-2 microglobulin, cystatin C, lipocalin-2 / NGAL) are quantified under panel 2 (MILLIPLEX® MAP Rat Kidney Toxicity Magnetic Bead Panel 2, RKTX2MAG-37K). The The assay for determining the concentration of these biomarkers in rat urine is based on Luminex xMAP® technology. Microspheres coated with anti-α-GST / β-2 microglobulin / calbindin / clusterin / cystatin C / KIM-1 / osteopontin / TIMP-1 / VEGF / NGAL antibody are color-coded with two different fluorescent dyes. The following parameters are determined (using urine ADVIA 1650): urinary protein, urinary creatinine. Quantitative parameters: volume, pH (using 10-Multistix SG test strip / Clinitek 500 urine analyzer), specific gravity (using refractometer). Semi-quantitative parameters (using 10-Multistix SG test strip / Clinitek 500 urine analyzer): protein, glucose, ketone, bilirubin, nitrite, blood, urobilinogen, sedimented cytology (by microscopy). Qualitative parameters: appearance, color. After sacrifice, body weight and kidney, liver and spleen weights are measured and the organ to body weight ratio is calculated. Kidney and liver samples are taken and frozen or stored in formalin. Perform microscopic analysis.

The present invention provides the use of an oligomeric compound of the invention for the preparation of a medicament for the treatment of a disease or disorder, eg a metabolic disease or disorder.
[Invention 1001]
The following three areas:
(I) a first region (region A) comprising 7 to 26 contiguous nucleotides;
(Ii) a second region comprising 1-10 nucleotides (region B) covalently linked to the 5 ′ nucleotide or 3 ′ nucleotide of the first region via, for example, an internucleoside linking group, such as a phosphodiester linkage. ),here,
(A) the internucleoside linkage between the first region and the second region is a phosphodiester linkage, and the nucleoside of the second region adjacent to the first region is either DNA or RNA; and / or
(B) a DNA or RNA nucleoside in which at least one nucleoside of the second region is linked by a phosphodiester;
(Iii) a third region comprising a conjugate moiety, targeting moiety, reactive group or blocking moiety and covalently linked to the second region
An oligomeric compound having a length of 8 to 35 nucleotides.
[Invention 1002]
The internucleoside linkage of the first region comprises at least one internucleoside linkage other than a phosphodiester, such as at least one of the internucleoside linkages within region A, such as at least 50%, such as at least 75%, such as The oligomer of the present invention 1001, wherein at least 90%, such as 100%, is other than a phosphodiester.
[Invention 1003]
The oligomer of invention 1001 or 1002, wherein the internucleoside linkage other than the phosphodiester is a sulfur-containing internucleoside linkage, such as phosphorothioate, phosphorodithioate, and boranophosphate, such as phosphorothioate.
[Invention 1004]
The oligomer according to any one of the inventions 1001 to 1003, wherein the first region is an antisense oligomer comprising a sequence complementary to the nucleic acid target.
[Invention 1005]
The oligomer of the present invention 1004, wherein the nucleic acid target is selected from the group consisting of microRNA and mRNA.
[Invention 1006]
The oligomer of any of the inventions 1001-1005, wherein the first region comprises at least one nucleoside analog.
[Invention 1007]
For example, the oligomer of the invention 1006, wherein the first region comprises a gapmer, a mixmer, or a totalmer.
[Invention 1008]
For example, the oligomer of the invention 1006 or 1007, wherein the first region comprises at least one bicyclic nucleotide analog (LNA).
[Invention 1009]
The oligomer of any of the invention 1001-1008, wherein the first region and the second region form a continuous nucleotide sequence.
[Invention 1010]
The oligomer of any of 1001 to 1009 of the invention, wherein the second region is 5 ′ of the first region.
[Invention 1011]
The oligomer according to any of the invention 1001 to 1009, wherein the second region is 3 ′ of the first region.
[Invention 1012]
The oligomer of any of the inventions 1001-1011, wherein the second region comprises at least 2, for example at least 3, consecutive nucleoside units selected from the group of DNA units and RNA units, which can be linked by phosphodiester linkages.
[Invention 1013]
The second region is covalently linked to the third region at the terminal nucleoside of the second region (ie, the 3 ′ end or 5 ′ end, depending on the position of the first region) of the invention 1001-1012 Any oligomer.
[Invention 1014]
The oligomer of any of the invention 1001-1013, wherein the third region comprises a non-nucleotide moiety, such as a conjugate group, such as a sterol, such as cholesterol, or a carbohydrate, such as a GalNac / GalNac cluster.
[Invention 1015]
The third region consists of lipophilic groups (eg lipids, fatty acids, sterols), proteins, peptides, antibodies or fragments thereof, polymers, reporter groups, dyes, receptor ligands, small molecule drugs, prodrugs, and vitamins The oligomer of the present invention 1014 comprising a more selected moiety.
[Invention 1016]
The oligomer of any of the inventions 1001 to 1013, wherein the third region comprises a nucleic acid or polymer thereof, such as an aptamer, or a blocking nucleotide sequence.
[Invention 1017]
The oligomer of any of 1001 to 1016 of the invention, wherein the third region comprises a targeting group.
[Invention 1018]
The oligomer of any of 1001 to 1013 of the present invention, wherein the third region comprises an activated group.
[Invention 1019]
The oligomer of any of 1001 to 1018 of the invention, wherein the second region and the third region are covalently joined by a linker group.
[Invention 1020]
The present invention 1001 wherein the linker group between the second region and the third region comprises a group selected from a phosphodiester group, a phosphorothioate group, a phosphorodithioate group, or a boranophosphate group, such as a phosphodiester group. Any oligomer of ~ 1019.
[Invention 1021]
A pharmaceutical composition comprising any oligomer compound of the present invention and a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.
[Invention 1022]
The oligomeric compound of any of the present invention for use in inhibiting a nucleic acid target in a cell.
[Invention 1023]
The oligomeric compound of any of the present invention for use in medicine.
[Invention 1024]
The oligomeric compound of any of the present invention for use in the treatment of a medical disease or disorder.
[Invention 1025]
Use of any oligomeric compound according to the invention as described above for the preparation of a medicament for the treatment of a disease or disorder, for example a metabolic disease or disorder.
[Invention 1026]
A method of synthesizing an oligomeric compound comprising a first region, a second region, and a third region, optionally comprising a linker region (Y), as defined in the present invention 1001-1025,
(A) One of the following [third area]:
(I) a linker group (-Y-),
(Ii) a group selected from the group consisting of conjugates, targeting groups, blocking groups, reactive groups [eg, amines or alcohols], or activating groups (X-),
(Iii) -YX group
Providing a [solid phase] oligonucleotide synthesis support to which is attached, and
(B) region B, followed by step [sequential] oligonucleotide synthesis of region A, and / or
(C) comprising a step of [sequential] oligonucleotide synthesis of the first region (A) and the second region (B), and following the synthesis step,
(D) the third region [including phosphoramidite],
(I) a linker group (-Y-),
(Ii) a group selected from the group consisting of conjugates, targeting groups, blocking groups, reactive groups [eg, amines or alcohols], or activating groups (X-),
(Iii) -YX group
Followed by the process of adding
(E) A step of cleaving the oligomer compound from the [solid phase] support
Including, optionally,
(F) the third group is an activating group and activates the activating group to produce a reactive group, followed by a conjugate group, blocking group, or targeting, optionally via a linker group (Y) Adding a group to the reactive group;
(G) the third region is a reactive group, optionally adding a conjugate group, a blocking group, or a targeting group to the reactive group via a linker group (Y);
(H) The third region is a linker group (Y), and a conjugate group, a blocking group, or a targeting group is added to the linker group (Y)
More steps to be selected
Further including
A method wherein step (f), (g), or (h) is performed either before or after cleavage of the oligomeric compound from the oligonucleotide synthesis support.
[Invention 1027]
A method of treating a disease or disorder in a subject in need of treatment comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the oligomeric compound of the invention.
[Invention 1028]
Appropriately inhibits expression of a target gene in a cell, comprising administering the oligomeric compound according to the present invention to the cell expressing the target gene appropriately in an amount effective to reduce the expression of the target gene in the cell Method.

Claims (28)

The following three areas:
(I) a first region (region A) comprising 7 to 26 contiguous nucleotides;
(Ii) a second region comprising 1-10 nucleotides (region B) covalently linked to the 5 ′ nucleotide or 3 ′ nucleotide of the first region via, for example, an internucleoside linking group, such as a phosphodiester linkage. ),here,
(A) the internucleoside linkage between the first region and the second region is a phosphodiester linkage, and the nucleoside of the second region adjacent to the first region is either DNA or RNA; and / or (b) ) A DNA or RNA nucleoside in which at least one nucleoside of the second region is linked by a phosphodiester;
(Iii) an 8-35 nucleotide long oligomeric compound comprising a third region comprising a conjugate moiety, a targeting moiety, a reactive group, or a blocking moiety and covalently linked to the second region.   The internucleoside linkage of the first region comprises at least one internucleoside linkage other than a phosphodiester, such as at least one of the internucleoside linkages within region A, such as at least 50%, such as at least 75%, such as 2. The oligomer of claim 1, wherein at least 90%, such as 100%, is other than a phosphodiester.   The oligomer according to claim 1 or 2, wherein the internucleoside linkage other than the phosphodiester is a sulfur-containing internucleoside linkage, such as phosphorothioate, phosphorodithioate, and boranophosphate, such as phosphorothioate.   The oligomer according to any one of claims 1 to 3, wherein the first region is an antisense oligomer comprising a sequence complementary to a nucleic acid target.   5. The oligomer according to claim 4, wherein the nucleic acid target is selected from the group consisting of microRNA and mRNA.   6. The oligomer according to any one of claims 1 to 5, wherein the first region comprises at least one nucleoside analogue.   For example, the oligomer of claim 6, wherein the first region comprises a gapmer, a mixmer, or a totalmer.   For example, the oligomer of claim 6 or 7, wherein the first region comprises at least one bicyclic nucleotide analog (LNA).   The oligomer according to any one of claims 1 to 8, wherein the first region and the second region form a continuous nucleotide sequence.   10. The oligomer according to any one of claims 1 to 9, wherein the second region is 5 'of the first region.   10. The oligomer according to any one of claims 1 to 9, wherein the second region is 3 'of the first region.   The second region comprises at least 2, for example at least 3, consecutive nucleoside units selected from the group of DNA units and RNA units, which can be linked by phosphodiester linkages. Oligomers.   13. The second region of claim 1-12, wherein the second region is covalently linked to the third region at a terminal nucleoside of the second region (i.e., 3 'or 5' end, depending on the position of the first region). The oligomer according to any one of the above.   14. The oligomer according to any one of claims 1 to 13, wherein the third region comprises a non-nucleotide moiety, such as a conjugate group, such as a sterol, such as cholesterol, or a carbohydrate, such as a GalNac / GalNac cluster.   The third region consists of lipophilic groups (eg lipids, fatty acids, sterols), proteins, peptides, antibodies or fragments thereof, polymers, reporter groups, dyes, receptor ligands, small molecule drugs, prodrugs, and vitamins 15. The oligomer of claim 14, comprising a more selected moiety.   14. The oligomer according to any one of claims 1 to 13, wherein the third region comprises a nucleic acid or a polymer thereof, such as an aptamer, or a blocking nucleotide sequence.   The oligomer according to any one of claims 1 to 16, wherein the third region comprises a targeting group.   14. The oligomer according to any one of claims 1 to 13, wherein the third region comprises an activated group.   The oligomer according to any one of claims 1 to 18, wherein the second region and the third region are covalently joined by a linker group.   The linker group between the second region and the third region comprises a group selected from a phosphodiester group, phosphorothioate group, phosphorodithioate group, or boranophosphate group, such as a phosphodiester group. 20. The oligomer according to any one of -19.   A pharmaceutical composition comprising an oligomeric compound according to any one of the preceding claims and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.   The oligomeric compound according to any one of the preceding claims for use in the inhibition of nucleic acid targets in cells.   The oligomeric compound according to any one of the preceding claims, for use in medicine.   An oligomeric compound according to any one of the preceding claims for use in the treatment of a medical disease or disorder.   Use of an oligomeric compound according to any of the preceding claims for the preparation of a medicament for the treatment of a disease or disorder, for example a metabolic disease or disorder. A method for synthesizing an oligomeric compound comprising a first region, a second region, and a third region, optionally comprising a linker region (Y), as defined in claims 1-25, comprising:
(A) One of the following [third area]:
(I) a linker group (-Y-),
(Ii) a group selected from the group consisting of conjugates, targeting groups, blocking groups, reactive groups [eg, amines or alcohols], or activating groups (X-),
(Iii) providing a [solid phase] oligonucleotide synthesis support to which a -YX group is attached, and (b) a step of region B followed by a [sequential] oligonucleotide synthesis of region A, and / or (C) comprising a step of [sequential] oligonucleotide synthesis of the first region (A) and the second region (B), and following the synthesis step,
(D) the third region [including phosphoramidite],
(I) a linker group (-Y-),
(Ii) a group selected from the group consisting of conjugates, targeting groups, blocking groups, reactive groups [eg, amines or alcohols], or activating groups (X-),
(Iii) adding a -YX group, followed by
(E) [solid phase] cleaving the oligomeric compound from the support, optionally,
(F) the third group is an activating group and activates the activating group to produce a reactive group, followed by a conjugate group, blocking group, or targeting, optionally via a linker group (Y) Adding a group to the reactive group;
(G) the third region is a reactive group, optionally adding a conjugate group, a blocking group, or a targeting group to the reactive group via a linker group (Y);
(H) the third region is a linker group (Y) and further comprises a further step selected from the step of adding a conjugate group, blocking group, or targeting group to the linker group (Y);
A method wherein step (f), (g), or (h) is performed either before or after cleavage of the oligomeric compound from the oligonucleotide synthesis support.   A method of treating a disease or disorder in a subject in need of treatment comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the oligomeric compound of the invention.   Appropriately inhibits expression of a target gene in a cell, comprising administering the oligomeric compound according to the present invention to the cell expressing the target gene appropriately in an amount effective to reduce the expression of the target gene in the cell Method.

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